Plant micrornas and methods of use thereof

ABSTRACT

This invention discloses novel microRNAs and their precursors, and recombinant DNA constructs including such novel miRNAs, miRNA precursors, miRNA promoters, and miRNA recognition sites corresponding to the miRNAs. Included are novel miRNA and miRNA precursors that exhibit nutrient-responsive expression. Also disclosed are miRNA decoy sequences. Further provided are non-natural transgenic plant cells, plants, and seeds containing in their genome a recombinant DNA construct of this invention and methods of controlling gene expression using recombinant DNA constructs of this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCELISTINGS

This application is a continuation of U.S. patent application Ser. No.11/974,469, filed on Oct. 12, 2007, which claims priority to U.S.Provisional Patent Application No. 60/851,187, filed on Oct. 12, 2006,U.S. Provisional Patent Application No. 60/908,826, filed Mar. 29, 2007,and U.S. Provisional Patent Application No. 60/969,195, filed Aug. 31,2007, all of which are incorporated by reference in their entiretyherein. A computer readable form of the Sequence Listing is filed withthis application by electronic submission and is incorporated into thisapplication by reference in its entirety. The Sequence Listing iscontained in the file created on Dec. 9, 2014, having the file nameP34154USO4 SL.txt, and is 2,265,088 bytes in size (as measured in theMS-Windows® operating system).

FIELD OF THE INVENTION

This invention discloses novel microRNAs and microRNA precursors,recombinant DNA constructs including such novel miRNAs, miRNAprecursors, and miRNA recognition sites corresponding to the miRNAs.Included are novel miRNA and miRNA precursors that exhibitabiotic-stress-responsive expression. Further provided are miRNA decoysequences, non-natural transgenic plant cells, plants, and seedscontaining in their genome a recombinant DNA construct of thisinvention, and methods of controlling gene expression using recombinantDNA constructs of this invention.

BACKGROUND OF THE INVENTION

Several cellular pathways involved in RNA-mediated gene suppression havebeen described, each distinguished by a characteristic pathway andspecific components. See, for example, the reviews by Brodersen andVoinnet (2006), Trends Genetics, 22:268-280, and Tomari and Zamore(2005) Genes & Dev., 19:517-529. The siRNA pathway involves thenon-phased cleavage of a double-stranded RNA (“RNA duplex”) to smallinterfering RNAs (siRNAs). The microRNA pathway involves microRNAs(miRNAs), non-protein coding RNAs generally of between about 19 to about25 nucleotides (commonly about 20-24 nucleotides in plants) that guidecleavage in trans of target transcripts, negatively regulating theexpression of genes involved in various regulation and developmentpathways. Plant miRNAs have been defined by a set of characteristicsincluding a stem-loop precursor that is processed by DCL1 to a singlespecific ˜21-nucleotide miRNA, expression of a single pair of miRNA andmiRNA* species from the RNA duplex with two-nucleotide 3′ overhangs, andsilencing of specific targets in trans. See Bartel (2004) Cell,116:281-297; Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385;Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol., 57:19-53; Ambros etal. (2003) RNA, 9:277-279. In the trans-acting siRNA (ta-siRNA) pathway,miRNAs serve to guide in-phase processing of siRNA primary transcriptsin a process that requires an RNA-dependent RNA polymerase forproduction of an RNA duplex; trans-acting siRNAs are defined by lack ofsecondary structure, an miRNA target site that initiates production ofdouble-stranded RNA, requirements of DCL4 and an RNA-dependent RNApolymerase (RDR6), and production of multiple perfectly phased˜21-nucleotide small RNAs with perfectly matched duplexes withtwo-nucleotide 3′ overhangs (see Allen et al. (2005) Cell, 121:207-221).

MicroRNAs (miRNAs) are non-protein coding RNAs, generally of betweenabout 19 to about 25 nucleotides (commonly about 20-24 nucleotides inplants), that guide cleavage in trans of target transcripts, negativelyregulating the expression of genes involved in various regulation anddevelopment pathways (Bartel (2004) Cell, 116:281-297). In some cases,miRNAs serve to guide in-phase processing of siRNA primary transcripts(see Allen et al. (2005) Cell, 121:207-221).

Some microRNA genes (MIR genes) have been identified and made publiclyavailable in a database (‘miRBase“, available on line atmicrorna.sanger.ac.uk/sequences). The applicants have disclosed novelMIR genes, mature miRNAs, and miRNA recognition sites in U.S. patentapplication Ser. No. 11/303,745, filed 15 Dec. 2005, which areincorporated by reference herein. Additional MIR genes and mature miRNAsare also described in U.S. Patent Application Publications 2005/0120415and 2005/144669A1, which are incorporated by reference herein. MIR geneshave been reported to occur in intergenic regions, both isolated and inclusters in the genome, but can also be located entirely or partiallywithin introns of other genes (both protein-coding andnon-protein-coding). For a recent review of miRNA biogenesis, see Kim(2005) Nature Rev. Mol. Cell Biol., 6:376-385. Transcription of MIRgenes can be, at least in some cases, under promotional control of a MIRgene's own promoter. MIR gene transcription is probably generallymediated by RNA polymerase II (see, e.g., Aukerman. and Sakai (2003)Plant Cell, 15:2730-2741; Parizotto et al. (2004) Genes Dev.,18:2237-2242), and therefore could be amenable to gene silencingapproaches that have been used in other polymerase II-transcribed genes.The primary transcript (which can be polycistronic) is termed a“pri-miRNA”, a miRNA precursor molecule that can be quite large (severalkilobases) and contains one or more local double-stranded or “hairpin”regions as well as the usual 5′ “cap” and polyadenylated tail of anmRNA. See, for example, FIG. 1 in Kim (2005) Nature Rev. Mol. CellBiol., 6:376-385.

In plant cells, microRNA precursor molecules are believed to be largelyprocessed in the nucleus. The pri-miRNA is processed to a shorter miRNAprecursor molecule that also includes a stem-loop or fold-back structureand is termed the “pre-miRNA”. In plants, miRNAs and siRNAs are formedby distinct DICER-like (DCL) enzymes, and in Arabidopsis a nuclear DCLenzyme (DCL1) is believed to be required for mature miRNA formation;see, for example, Ambros et al. (2003) RNA, 9:277-279, and Xie et al.(2004) PLoS Biol., 2:642-652. Additional reviews on microRNA biogenesisand function are found, for example, in Bartel (2004) Cell, 116:281-297;Murchison and Hannon (2004) Curr. Opin. Cell Biol., 16:223-229; andDugas and Bartel (2004) Curr. Opin. Plant Biol., 7:512-520. MicroRNAscan thus be described in terms of RNA (e.g., RNA sequence of a maturemiRNA or a miRNA precursor RNA molecule), or in terms of DNA (e.g., DNAsequence corresponding to a mature miRNA RNA sequence or DNA sequenceencoding a MIR gene or fragment of a MIR gene or a miRNA precursor).

MIR gene families are estimated to account for 1% of at least somegenomes and capable of influencing or regulating expression of about athird of all genes (see, e.g., Tomari et al. (2005) Curr. Biol.,15:R61-64; G. Tang (2005) Trends Biochem. Sci., 30:106-14; Kim (2005)Nature Rev. Mol. Cell Biol., 6:376-385). Because miRNAs are importantregulatory elements in eukaryotes, including animals and plants,transgenic suppression of miRNAs could, for example, lead to theunderstanding of important biological processes or allow themanipulation of certain pathways (e.g., regulation of cellulardifferentiation, proliferation, and apoptosis) useful, for example, inbiotechnological applications. See, for example, O'Donnell et al. (2005)Nature, 435:839-843; Cai et al. (2005) Proc. Natl. Acad. Sci. USA,102:5570-5575; Morris and McManus (2005) Sci. STKE, pe41(stke.sciencemag.org/cgi/reprint/sigtrans;2005/297/pe41.pdf). MicroRNA(MIR) genes have identifying characteristics, including conservationamong plant species, a stable foldback structure, and processing of aspecific miRNA/miRNA* duplex by Dicer-like enzymes (Ambros et al. (2003)RNA, 9:277-279). These characteristics have been used to identify miRNAsand their corresponding genes in plants (Xie et al. (2005) PlantPhysiol., 138:2145-2154; Jones-Rhoades and Bartel (2004) Mol. Cell,14:787-799; Reinhart et al. (2002) Genes Dev., 16:1616-1626; Sunkar andZhu (2004) Plant Cell, 16:2001-2019). Publicly available microRNA genesare catalogued at miRBase (Griffiths-Jones et al. (2003) Nucleic AcidsRes., 31:439-441).

MiRNAs are expressed in very specific cell types in Arabidopsis (see,for example, Kidner and Martienssen (2004) Nature, 428:81-84, Millar andGubler (2005) Plant Cell, 17:705-721). Suppression can be limited to aside, edge, or other division between cell types, and is believed to berequired for proper cell type patterning and specification (see, e.g.,Palatnik et al. (2003) Nature, 425:257-263). Suppression of a GFPreporter gene containing an endogenous miR171 recognition site was foundto limit expression to specific cells in transgenic Arabidopsis(Parizotto et al. (2004) Genes Dev., 18:2237-2242). Recognition sites ofmiRNAs have been validated in all regions of an mRNA, including the 5′untranslated region, coding region, and 3′ untranslated region,indicating that the position of the miRNA target site relative to thecoding sequence may not necessarily affect suppression (see, e.g.,Jones-Rhoades and Bartel (2004). Mol. Cell, 14:787-799, Rhoades et al.(2002) Cell, 110:513-520, Allen et al. (2004) Nat. Genet., 36:1282-1290,Sunkar and Zhu (2004) Plant Cell, 16:2001-2019).

The mature miRNAs disclosed herein are processed from MIR genes thatgenerally belong to canonical families conserved across distantlyrelated plant species. These MIR genes and their encoded mature miRNAsare also useful, e.g., for modifying developmental pathways, e.g., byaffecting cell differentiation or morphogenesis (see, for example,Palatnik et al. (2003) Nature, 425:257-263; Mallory et al. (2004) Curr.Biol., 14:1035-1046), to serve as sequence sources for engineered(non-naturally occurring) miRNAs that are designed to silence sequencesother than the transcripts targeted by the naturally occurring miRNAsequence (see, for example, Parizotto et al. (2004) Genes Dev.,18:2237-2242; also see U.S. Patent Application Publications 2004/3411A1and 2005/0120415, incorporated by reference herein), and to stabilizedsRNA. A MIR gene itself (or its native 5′ or 3′ untranslated regions,or its native promoter or other elements involved in its transcription)is useful as a target gene for gene suppression (e.g., by methods of thepresent invention), where suppression of the miRNA encoded by the MIRgene is desired. Promoters of MIR genes can have very specificexpression patterns (e.g., cell-specific, tissue-specific, or temporallyspecific), and thus are useful in recombinant constructs to induce suchspecific transcription of a DNA sequence to which they are operablylinked.

This invention provides novel microRNAs and microRNA precursorsidentified from plants (including crop plants such as maize, rice, andsoybean), as well as recombinant DNA constructs including such novelmiRNAs, miRNA precursors, miRNA recognition sites, miRNA decoysequences, and miRNA promoters corresponding to the miRNAs. Alsodisclosed and claimed are non-natural transgenic plant cells, plants,and seeds containing in their genome a recombinant DNA construct of thisinvention. Further provided are methods of gene suppression usingrecombinant DNA constructs of this invention and methods of providingtransgenic plants with desired phenotypes, especially transgenic plantsexhibiting increased yield (relative to non-transgenic plants) underabiotic stress conditions including drought, nutrient deficiency, andcold or heat stress.

SUMMARY OF THE INVENTION

In one aspect, this invention provides a recombinant DNA constructincluding at least one transcribable DNA element for modulating theexpression of at least one target gene, wherein the at least onetranscribable DNA element is selected from the group consisting of: (a)a DNA element that transcribes to an miRNA precursor with the fold-backstructure of a plant miRNA precursor sequence selected from SEQ ID NOS.1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS.8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819,wherein the miRNA precursor includes a contiguous segment of at least90% of the nucleotides of the maize, rice, or soybean miRNA precursorsequence; (b) a DNA element that transcribes to an engineered miRNAprecursor derived from the fold-back structure of a plant miRNAprecursor sequence selected from SEQ ID NOS. 1036-2690, SEQ ID NOS.3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO.8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819, wherein the engineeredmiRNA precursor includes a modified mature miRNA; (c) a DNA element thatis located within or adjacent to a transgene transcription unit and thatis transcribed to RNA including a miRNA recognition site recognized by amature miRNA selected from SEQ ID NOS. 1-1035, SEQ ID NOS. 2730-3921,SEQ ID NOS. 5498-6683, SEQ ID NOS. 8409-8560, SEQ ID NO 8742, SEQ ID NO.8744, SEQ ID NOS. 8812-8815, SEQ ID NO. 8845, and SEQ ID NO. 8850, or bya mature miRNA derived from a plant miRNA precursor sequence selectedfrom SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS.6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, andSEQ ID NOS. 8816-8819; and (d) a DNA element for suppressing expressionof an endogenous miRNA derived from a plant miRNA precursor sequenceselected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS.6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, andSEQ ID NOS. 8816-8819.

Another aspect of this invention provides a non-natural transgenic plantcell including any of the recombinant DNA constructs of this invention.Further provided is a non-natural transgenic plant containing thenon-natural transgenic plant cell of this invention, including plants ofany developmental stage, and including a regenerated plant prepared fromthe non-natural transgenic plant cells disclosed herein, or a progenyplant (which can be an inbred or hybrid progeny plant) of theregenerated plant, or seed of such a non-natural transgenic plant. Alsoprovided and claimed is a transgenic seed having in its genome any ofthe recombinant DNA constructs provided by this invention.

In a further aspect, this invention provides a method of effecting genesuppression, including the steps of: (a) providing a non-naturaltransgenic plant including a regenerated plant prepared from anon-natural transgenic plant cell of this invention, or a progeny plantof the regenerated plant; and (b) transcribing the recombinant DNAconstruct in the non-natural transgenic plant; wherein the transcribingproduces RNA that is capable of suppressing the at least one target genein the non-natural transgenic plant, and whereby the at least one targetgene is suppressed relative to its expression in the absence oftranscription of the recombinant DNA construct.

In yet another aspect, this invention provides a method of concurrentlyeffecting gene suppression of at least one target gene and geneexpression of at least one gene of interest, including the steps of: (a)providing a non-natural transgenic plant including a regenerated plantprepared from the non-natural transgenic plant cell of this invention,or a progeny plant of the regenerated plant, wherein the recombinant DNAconstruct further includes a gene expression element for expressing theat least one gene of interest; and (b) transcribing the recombinant DNAconstruct in the non-natural transgenic plant, wherein, when therecombinant DNA construct is transcribed in the non-natural transgenicplant, transcribed RNA that is capable of suppressing the at least onetarget gene and transcribed RNA encoding the at least one gene ofinterest are produced, whereby the at least one target gene issuppressed relative to its expression in the absence of transcription ofthe recombinant DNA construct and the at least one gene of interest isconcurrently expressed.

In a further aspect, this invention provides a recombinant DNA constructincluding a synthetic miRNA-unresponsive transgene sequence that isunresponsive to a given mature miRNA, wherein the syntheticmiRNA-unresponsive transgene sequence is: (a) derived from a nativelymiRNA-responsive sequence by deletion or modification of all nativemiRNA recognition sites recognized by the given mature miRNA within thenatively miRNA-responsive sequence, and (b) is not recognized by thegiven mature miRNA.

In another aspect, this invention provides a recombinant DNA constructincluding a promoter of a miRNA that exhibits an expression pattern thatis responsive to abiotic stress, for example, a promoter of a miRNA thatexhibits an expression pattern characterized by suppression of the miRNAunder nutrient stress, a promoter of a miRNA that exhibits an expressionpattern characterized by suppression of the miRNA under water stress, ora promoter of a miRNA that exhibits an expression pattern characterizedby suppression of the miRNA under temperature stress.

In still a further aspect, this invention provides a recombinant DNAconstruct that is transcribed to an RNA transcript including at leastone miRNA decoy sequence that is recognized and bound by an endogenousmature miRNA but not cleaved; included are transgenic plant cells,plants, and seeds having this construct in their genome, and methods ofuse of this construct. Related aspects of this invention includerecombinant DNA constructs and methods for suppression of endogenousmiRNA decoy sequences. Also disclosed are analogous decoy sequences thatrecognize and bind to other small RNAs (ta-siRNAs, nat-siRNAs, andphased small RNAs) but are not cleaved, thus reducing the activity ofthe small RNA.

Other specific embodiments of the invention are disclosed in thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a non-limiting example of a fold-back structure of aplant miRNA precursor sequence selected from SEQ ID NOS. 1036-2690, SEQID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ IDNO. 8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819, more specifically,the fold-back structure of the miRNA precursor sequence having SEQ IDNO. 1136, which includes two short stem-loops, a loop, and two bulges.The miRNA precursor is processed in planta to a mature miRNA (in thisparticular example, to the mature miRNA having SEQ ID NO. 32).

FIGS. 2 and 3 depict non-limiting examples of DNA elements forsuppressing expression of a target gene, e.g., an endogenous miRNA, asdescribed in Example 3.

FIG. 4 depicts Northern blot results for mature miRNAs isolated fromdifferent maize tissues, as described in Example 4.

FIG. 5 depicts transcription profiles of probeset sequences includingmiRNA precursor sequences having expression patterns specific to maizemale reproductive tissue (pollen), as described in Example 4.

FIG. 6 depicts drought stages for soybean plants a relative scoringsystem from 1.0 (no effect or control) to 4.0, as described in Example5.

FIG. 7 depicts the fold-back structures of miRNA precursors fromdifferent plants, as described in Example 6. Panel A depicts thefold-back structure of a miRMON18 precursor from maize (SEQ ID NO.3936), Panel B depicts the fold-back structure of a miRMON18 precursorfrom rice (SEQ ID NO. 1763), and Panel C depicts the fold-back structureof a miR827 precursor from Arabidopsis thaliana (SEQ ID NO. 8743). PanelD depicts a comparison of miR827 (SEQ ID NO. 8744) and miRMON18 (SEQ IDNO. 393, SEQ ID NO. 3227, or SEQ ID NO. 8742), with numbered arrowsindicating positions 1, 10, and 21 of the mature miRNA; the nucleotideat position 10 is also underlined.

FIG. 8 depicts expression patterns of miRMON18 as determined by Northernblots of the mature miRMON18 21-mer (Panel A) and transcriptionprofiling of the miRMON18 precursor (Panel B), as described in Example6.

FIG. 9 depicts analysis of expression of the maize miRMON18 precursor(SEQ ID NO. 3936) in maize tissues from plants grown underwater-deficient (drought) (Panel A), cold (Panel B), andnitrogen-deficient conditions (Panel C), as described in Example 6.

FIG. 10 depicts results of northern blots of small RNAs in maize (Zeamays var. LH244), showing enhanced miRMON18 expression in maizeendosperm and kernel, and strong miRMON18 suppression in leaves inducedby nitrogen deficiency (Panel A), and strong miRMON18 expression in leaftissue under phosphate-sufficient conditions and miRMON18 suppressionunder phosphate-deficient conditions (Panel B), as described in Example6.

FIG. 11 depicts a multiple sequence alignment of novel maize miRMON18target genes containing the maize SPX domain (indicated by underlinedsequence, where present) and the maize MFS domain (indicated by sequencein bold text), as described in Example 7.

FIG. 12 depicts a phylogenetic tree constructed for the identified SPXgenes, as described in Example 7; genes containing a predicted miRMON18recognition site (in genes from species other than Arabidopsis thaliana)or a predicted miR827 recognition site (in genes from Arabidopsisthaliana) that has been experimentally validated are indicated in boldtext.

FIG. 13 depicts a miRMON18 genomic sequence (SEQ ID NO. 8800), asdescribed in Example 8. This shows the miRMON18 transcript in upper-casetext at nucleotides 2173-2788 a miRMON18 promoter element in lower-casetext at nucleotides 211-2172, a leader element in lower-case text atnucleotides 2173-2308, a canonical TATA box (ending 25 nucleotidesupstream of the transcription start site) in underlined lower-case textat nucleotides 2144-2147, the mature miRMON18 as underlined upper-casetext at nucleotides 2419-2439, and the miRMON18* as underlinedupper-case text at nucleotides 2322-2341.

FIG. 14 depicts the predicted cleavage by miRMON18 of the rice sequencesOs02g45520 (SEQ ID NO. 8784) and Os04g48390 (SEQ ID NO. 8786) and themaize sequence MRT4577_(—)36529C (SEQ ID NO. 8788), as described inExample 9.

FIG. 15 depicts the inverse correlation between the miRMON18 precursor(Panel A) and a miRMON18 target (Panel B), as described in Example 9.Panel B shows that the maize sequence MRT4577_(—)36529C (SEQ ID NO.8788), exhibited higher expression levels under nitrogen-deficientconditions than under nitrogen-sufficient conditions, i.e., anexpression pattern opposite to that of the miRMON18 precursor as shownin Panel A.

FIG. 16 depicts the vector pMON107261, which includes a CaMV 35Spromoter driving expression of the maize miRMON18 transcript (e.g.,nucleotides 2173-2788 of SEQ ID NO. 8800), as described in Example 10.

FIG. 17 depicts the fold-back structures of maize miR399 precursors(Panel A) and results of transcriptional profiling experiments (PanelB), which demonstrate that the Zm-miR399 pri-miRNA is suppressed undernitrogen-deficient conditions (black bars) and is expressed undernitrogen-sufficient conditions (white bars), as described in Example 11.

FIG. 18 depicts alignment of the maize cDNA sequences of the miR399decoy sequences, with the consensus sequence given as SEQ ID NO. 8834,and reveals at least two groups of genes containing miR399 decoysequences, as described in Example 11.

FIG. 19 depicts experiments comparing expression of maize miR399 decoysequences and miR399 precursors as described in Example 11. Panel Ashows a transcription profile of group 1 miR399 decoy geneMRT4577_(—)47862C.7 (SEQ ID NO. 8827) and Panel B shows a transcriptionprofile of group 2 miR399 decoy gene MRT4577_(—)36567C.8 (SEQ ID NO.8829), indicating that these miR399 decoy sequences are down-regulatedby nitrogen deficiency. These results were verified by northern blotsmeasuring expression of the mature miR399 (Panel C) and of the miR399decoy sequence MRT4577_(—)47862C.7 (SEQ ID NO. 8827) (Panel D).

FIG. 20 depicts transcription profiling experiments comparing expressionof maize endogenous miR399 decoy cDNA sequences and the correspondingmaize miR399 precursors under different temperature conditions, asdescribed in Example 11. Group 2 miR399 decoy gene MRT4577_(—)36567C.8(SEQ ID NO. 8829) exhibited at least ten-fold or greater higherexpression during nitrogen-sufficient conditions in maize leaf,especially during daylight hours (Panel A). This same gene exhibited atleast a two-fold down-regulation in root (Panel B) and in shoot (PanelC) after extended exposure to cold.

FIG. 21 depicts expression of endogenous miR399 decoy cDNA sequences indifferent tissues in both maize and soybean, as described in Example 11.Panel A depicts expression levels of the group 1 maize miR399 decoysequence SEQ ID NO. 8827 (MRT4577_(—)47862C, represented by probesA1ZMO05814_at and A1ZMO05813_s_at), and the group 2 maize miR399 decoysequence SEQ ID NO. 8829 (MRT4577_(—)36567C, represented by probeA1ZM048024_at), as well as of the maize pri-miR399 sequence SEQ ID NO.8818 (MRT4577_(—)22487C.6 represented by probe A1ZM033468_at). Panel Bdepicts expression levels of the soybean miR399 decoy sequences SEQ IDNO. 8842 (MRT3847_(—)217257C.2, represented by probe A1GM031412_at), SEQID NO. 8844 (MRT3847_(—)236871C.2, represented by probe A1GM053788_at),SEQ ID NO. 8836 (MRT3847_(—)238967C.1, represented by probeA1GM035741_at), and SEQ ID NO. 8838 (MRT3847_(—)241832C.1, representedby probe A1GM069937_at).

FIG. 22 depicts transcription profiling data in various soybean tissuesof the soybean endogenous miR319 decoy SEQ ID NO. 8847(MRT3847_(—)41831C.6, represented by probe A1GM001017 at) (Panel A) andtranscription profiling data in various maize tissues of the maizeendogenous miR319 decoy SEQ ID NO. 8849 (MRT4577_(—)577703C.1,represented by probe A1ZMO12886_s_at) (Panel B), as described in Example11.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Generally, the nomenclature usedand the manufacture or laboratory procedures described below are wellknown and commonly employed in the art. Conventional methods are usedfor these procedures, such as those provided in the art and variousgeneral references. Unless otherwise stated, nucleic acid sequences inthe text of this specification are given, when read from left to right,in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNAor as RNA, as specified; disclosure of one necessarily defines theother, as is known to one of ordinary skill in the art. Where a term isprovided in the singular, the inventors also contemplate aspects of theinvention described by the plural of that term. The nomenclature usedand the laboratory procedures described below are those well known andcommonly employed in the art. Where there are discrepancies in terms anddefinitions used in references that are incorporated by reference, theterms used in this application shall have the definitions given. Othertechnical terms used have their ordinary meaning in the art that theyare used, as exemplified by a variety of technical dictionaries. Theinventors do not intend to be limited to a mechanism or mode of action.Reference thereto is provided for illustrative purposes only.

Recombinant DNA Constructs

This invention provides a recombinant DNA construct including at leastone transcribable DNA element for modulating the expression of at leastone target gene, wherein the at least one transcribable DNA element isselected from the group consisting of: (a) a DNA element thattranscribes to an miRNA precursor with the fold-back structure of aplant miRNA precursor sequence selected from SEQ ID NOS. 1036-2690, SEQID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ IDNO. 8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819, wherein the miRNAprecursor includes a contiguous segment of at least 90% of thenucleotides of the maize, rice, or soybean miRNA precursor sequence; (b)a DNA element that transcribes to an engineered miRNA precursor derivedfrom the fold-back structure of a plant miRNA precursor sequenceselected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS.6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, andSEQ ID NOS. 8816-8819, wherein the engineered miRNA precursor includes amodified mature miRNA; (c) a DNA element that is located within oradjacent to a transgene transcription unit and that is transcribed toRNA including a miRNA recognition site recognized by a mature miRNAselected from a mature miRNA selected from SEQ ID NOS. 1-1035, SEQ IDNOS. 2730-3921, SEQ ID NOS. 5498-6683, SEQ ID NOS. 8409-8560, SEQ ID NO8742, SEQ ID NO. 8744, SEQ ID NOS. 8812-8815, SEQ ID NO. 8845, and SEQID NO. 8850, or by a mature miRNA derived from a plant miRNA precursorsequence selected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO.8800, and SEQ ID NOS. 8816-8819; and (d) a DNA element for suppressingexpression of an endogenous miRNA derived from a plant miRNA precursorsequence selected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO.8800, and SEQ ID NOS. 8816-8819. Target genes, the expression of whichcan be modulated by use of a recombinant DNA construct of thisinvention, are described under the heading “Target Genes”. Embodimentsand utilities of the at least one transcribable DNA element aredescribed below.

(A) Expression of a Native miRNA Under Non-Native Conditions.

In one embodiment of the recombinant DNA construct, the at least onetranscribable DNA element for modulating the expression of at least onetarget gene includes a DNA element that transcribes to an miRNAprecursor with the fold-back structure of a plant miRNA precursorsequence selected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO.8800, and SEQ ID NOS. 8816-8819, wherein the miRNA precursor includes acontiguous segment of at least 90% of the nucleotides of the maize,rice, or soybean miRNA precursor sequence. In preferred embodiments, theat least one target gene is an endogenous gene of a plant, andexpression of the recombinant DNA construct in the plant results insuppression of the at least one target gene. By “miRNA precursor” ismeant a transcribed RNA that is larger than a mature miRNA processedfrom the miRNA precursor, and that typically can be predicted to form afold-back structure containing non-perfectly complementarydouble-stranded RNA regions. See Bartel (2004) Cell, 116:281-297; Kim(2005) Nature Rev. Mol. Cell Biol., 6:376-385; Jones-Rhoades et al.(2006) Annu. Rev. Plant Biol., 57:19-53; Ambros et al. (2003) RNA,9:277-279. Examples of microRNA precursors include, but are not limitedto, the primary miRNA transcript (pri-miRNA) as well as the pre-miRNAthat is natively derived from a pri-miRNA; miRNA precursors also includenon-natural RNA sequences that are predicted to form a fold-backstructure containing non-perfectly complementary double-stranded RNAregions and are processed in vivo, generally by one or more cleavagesteps, to a mature miRNA. By “miRNA precursor sequence” is meant an RNAsequence that includes at least the nucleotides of the miRNA precursorbut that may include additional nucleotides (such that the miRNAprecursor includes a contiguous segment of at least 90% of thenucleotides of the maize, rice, or soybean miRNA precursor sequence).Each miRNA precursor itself forms a fold-back structure that isidentical or near-identical to the fold-back structure that is formed byat least part of the corresponding miRNA precursor sequence.

In these embodiments, the miRNA precursor need not include all of thenucleotides contained in a plant miRNA precursor sequence selected fromSEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, and SEQ ID NOS.8816-8819, but preferably includes a contiguous segment of at least 80%,or at least 85%, or at least 90%, or at least 95%, or at least 97%, orat least 98%, or at least 99% of the nucleotides of a plant miRNAprecursor sequence selected from SEQ ID NOS. 1036-2690, SEQ ID NOS.3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO.8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819.

In preferred embodiments, the at least one target gene is an endogenousgene of a plant, and thus expression of the recombinant DNA construct inthe plant results in suppression of the at least one target gene.Transcription of the recombinant DNA construct in a transgenic plantcell modulates the expression of any gene (endogenous genes ortransgenes) that contains a sequence (“miRNA recognition site”) that issubstantially complementary to and recognized by the mature miRNAencoded by the miRNA precursor. Generally, transcription of therecombinant DNA construct results in suppression of an endogenous genethat contains a miRNA recognition site that is recognized by the maturemiRNA encoded by the miRNA precursor. In preferred embodiments, therecombinant DNA construct further includes a promoter other than thenative promoter of the miRNA sequence. This permits expression of themature miRNA under spatial or temporal or inducible conditions underwhich it would not natively be expressed. For example, the recombinantDNA construct can be designed to include a constitutive promoter andthus constitutively express a mature miRNA that is natively expressed(i.e., when expressed in the form of the endogenous miRNA precursorunder the control of the native promoter) only under dark conditions.Promoters that are useful with this recombinant DNA construct aredescribed under the heading “Promoters”.

In one non-limiting example, the recombinant DNA construct includes atranscribable DNA element for modulating the expression of at least onetarget gene, wherein the at least one transcribable DNA element includesa DNA element that transcribes to an miRNA precursor that is acontiguous segment consisting of about 90% of the nucleotides of themaize miRNA precursor sequence having SEQ ID NO. 1136, and that ispredicted to have a fold-back structure that is substantially the same(that is, having areas of double-stranded RNA stems and single-strandedloops or bulges in the same or approximately the same location) as thefold-back structure of the miRNA precursor sequence having SEQ ID NO.1136. The fold-back structure of the miRNA precursor sequence having SEQID NO. 1136 includes about 118 nucleotides, with two short stem-loopsprojecting from a loop at the closed end of the fold-back structure, andtwo small bulges within the main double-stranded “stem” of the fold-backstructure (FIG. 1). The mature miRNA processed in planta from a miRNAprecursor that is a contiguous segment consisting of about 90% of thenucleotides of the maize miRNA precursor sequence having SEQ ID NO. 1136is preferably identical to that encoded by the fold-back structure ofthe miRNA precursor sequence having SEQ ID NO. 1136, i.e., the maturemiRNA having SEQ ID NO. 32. Transcription of this recombinant DNAconstruct preferably results in suppression of at least one endogenousgene that contains a miRNA recognition site that is recognized by themature miRNA having SEQ ID NO. 32. While the maize miRNA precursorsequence having SEQ ID NO. 1136 is natively expressed in kernel tissuebut not in leaf (see Table 2), the recombinant DNA construct can furtherinclude a promoter other than the native promoter of the miRNA sprecursor sequence having SEQ ID NO. 1136, e.g., a constitutivepromoter, to allow transcription of a mature miRNA having SEQ ID NO. 32in tissues in addition to kernel tissue.

(B) Expression of an Engineered Mature miRNA.

In another embodiment of the recombinant DNA construct, the at least onetranscribable DNA element for modulating the expression of at least onetarget gene includes a DNA element that transcribes to an engineeredmiRNA precursor derived from the fold-back structure of a plant miRNAprecursor sequence selected from SEQ ID NOS. 1036-2690, SEQ ID NOS.3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO.8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819, wherein the engineeredmiRNA precursor includes a modified mature miRNA. In preferredembodiments, the at least one target gene is an endogenous gene of aplant or an endogenous gene of a pest or pathogen of the plant, andexpression of the recombinant DNA construct in the plant results insuppression of the at least one target gene. By “engineered” is meantthat nucleotides are changed (substituted, deleted, or added) in anative miRNA precursor sequence such a plant miRNA precursor sequenceselected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS.6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, andSEQ ID NOS. 8816-8819, thereby resulting in an engineered miRNAprecursor having substantially the same the fold-back structure as thenative miRNA precursor sequence, but wherein the mature miRNA that isprocessed from the engineered miRNA precursor has a modified sequence(i.e., different from that of the native mature miRNA) that is designedto suppress a target gene different from the target genes nativelysuppressed by the native miRNA precursor sequence.

One general, non-limiting method for determining nucleotide changes inthe native miRNA precursor sequence to produce the engineered miRNAprecursor, useful in making a recombinant DNA construct of thisinvention, includes the steps:

-   -   (a) Selecting a unique target sequence of at least 18        nucleotides specific to the target gene, e.g. by using sequence        alignment tools such as BLAST (see, for example, Altschul et        al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997)        Nucleic Acids Res., 25:3389-3402), for example, of both maize        cDNA and genomic DNA databases, to identify target transcript        orthologues and any potential matches to unrelated genes,        thereby avoiding unintentional silencing of non-target        sequences.    -   (b) Analyzing the target gene for undesirable sequences (e.g.,        matches to sequences from non-target species, especially        animals), and score each potential 19-mer segment for GC        content, Reynolds score (see Reynolds et al. (2004) Nature        Biotechnol., 22:326-330), and functional asymmetry characterized        by a negative difference in free energy (“ΔΔG”) (see Khvorova et        al. (2003) Cell, 115:209-216). Preferably 19-mers are selected        that have all or most of the following characteristics: (1) a        Reynolds score >4, (2) a GC content between about 40% to about        60%, (3) a negative ΔΔG, (4) a terminal adenosine, (5) lack of a        consecutive run of 4 or more of the same nucleotide; (6) a        location near the 3′ terminus of the target gene; (7) minimal        differences from the miRNA precursor transcript. Preferably        multiple (3 or more) 19-mers are selected for testing.    -   (c) Determining the reverse complement of the selected 19-mers        to use in making a modified mature miRNA; the additional        nucleotide at position 20 is preferably matched to the selected        target sequence, and the nucleotide at position 21 is preferably        chosen to be unpaired to prevent spreading of silencing on the        target transcript.    -   (d) Testing the engineered miRNA precursor, for example, in an        Agrobacterium mediated transient Nicotiana benthamiana assay for        modified mature miRNA expression and target repression.    -   and (e) Cloning the most effective engineered miRNA precursor        into a construct for stable transformation of maize (see the        sections under the headings “Making and Using Recombinant DNA        Constructs” and “Making and Using Non-natural Transgenic plant        Cells and Non-natural Transgenic Plants”).        (C) Expression of a Transgene and a miRNA Recognition Site.

In another embodiment of the recombinant DNA construct, the recombinantDNA construct further includes a transgene transcription unit, whereinthe at least one transcribable DNA element for modulating the expressionof at least one target gene includes a DNA element that is locatedwithin or adjacent to the transgene transcription unit and that istranscribed to RNA including a miRNA recognition site recognized by amature miRNA derived from a plant miRNA precursor sequence selected fromSEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, and SEQ ID NOS.8816-8819, and the at least one target gene includes the transgeneencoded by the transgene transcription unit, and wherein expression ofthe recombinant DNA construct in a plant results in expression of thetransgene in cells of the plant wherein the mature miRNA is not nativelyexpressed. Preferred embodiments of miRNA recognition sites are thosepredicted to be recognized by at least one mature miRNA selected from amature miRNA selected from SEQ ID NOS. 1-1035, SEQ ID NOS. 2730-3921,SEQ ID NOS. 5498-6683, SEQ ID NOS. 8409-8560, SEQ ID NO 8742, SEQ ID NO.8744, SEQ ID NOS. 8812-8815, SEQ ID NO. 8845, and SEQ ID NO. 8850, or byat least one mature miRNA derived from a plant miRNA precursor sequenceselected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS.6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, andSEQ ID NOS. 8816-8819. Prediction of a recognition site is achievedusing methods known in the art, such as sequence complementarity rulesas described by Zhang (2005) Nucleic Acids Res., 33:W701-704 and byRhoades et al. (2002) Cell, 110:513-520.

Prediction of a miRNA recognition site permits identification andvalidation of endogenous genes regulated by miRNAs from a nativelyexpressed miRNA precursor; this is useful, e.g., to eliminate or modifya miRNA recognition site in an endogenous gene in order to decoupleexpression of that gene from regulation by the endogenous miRNA thatnatively regulates expression of the gene. For example, the number ofmispairs involving bases at positions 2 to 13 (in a miRNA recognitionsite having contiguous 21 nucleotides) can be increased to preventrecognition and cleavage by the miRNA.

These recombinant DNA constructs are particularly useful for in plantaexpression of the transgene under a specific spatial, temporal, orinducible pattern without the need of a promoter having that specificexpression pattern. These recombinant DNA constructs allow, for example,the restricted expression of a gene transcribed by a constitutivepromoter or a promoter with expression beyond the desired cell or tissuetype(s). Restricted expression may be spatially or temporallyrestricted, e.g., restricted to specific tissues or cell types or files,or to specific developmental, reproductive, growth, or seasonal stages.Where a miRNA is expressed under particular conditions (e.g., underbiotic stress such as crowding, allelopathic interactions or pest orpathogen infestation, or abiotic stress such as heat or cold stress,drought stress, nutrient stress, heavy metal or salt stress), thecorresponding miRNA recognition site can be used for conditionallyspecific suppression, i.e., to suppress a transgene under the particularcondition. In a non-limiting example, a recombinant DNA construct ofthis invention that encodes (a) a transgene under the control of aconstitutive promoter and (b) a miRNA recognition site recognized by amature miRNA that is specifically expressed only under conditions ofwater stress, can be used for expression of the transgene in a plantunder non-water-stress conditions. In another non-limiting example, arecombinant DNA construct of this invention that encodes (a) a transgeneexpressing an insecticidal protein under the control of a promoterspecifically inducible by wounding, and (b) a miRNA recognition siterecognized by a mature miRNA that is expressed in tissues other thanroot, can be used for limited expression of the insecticidal protein inplant roots under conditions when the plant is wounded by an insectpest.

The transgene transcription unit includes at least a transgene, andoptionally additional sequence such as, but not limited to, a promoter,a promoter enhancer, a terminator, messenger RNA stabilizing ordestabilizing sequence (see, e.g., Newman et al. (1993) Plant Cell,5:701-714; Green (1993) Plant Physiol., 102:1065-1070; and Ohme-Takagiet al. (1993) Proc. Natl. Acad. Sci. USA, 90:11811-11815), sequence forlocalization or transport of the transgene transcript to a specificlocale (e.g., mitochondrion, plastid, nucleolus, peroxisome, endoplasmicreticulum, etc.), or other sequence related to the desired processing ofthe transgene. The transgene encoded by the transgene transcription unitcan include any one or more genes of interest, including codingsequence, non-coding sequence, or both. Genes of interest can includeany of the genes listed under “Target Genes”, preferred examples ofwhich include translatable (coding) sequence for genes encodingtranscription factors and genes encoding enzymes involved in thebiosynthesis or catabolism of molecules of interest (such as, but notlimited to, amino acids, fatty acids and other lipids, sugars and othercarbohydrates, biological polymers, and secondary metabolites includingalkaloids, terpenoids, polyketides, non-ribosomal peptides, andsecondary metabolites of mixed biosynthetic origin).

(D) Suppression of an Endogenous or Native miRNA.

In yet another embodiment of the recombinant DNA construct, the at leastone transcribable DNA element for modulating the expression of at leastone target gene includes a DNA element for suppressing expression of anendogenous miRNA derived from a plant miRNA precursor sequence selectedfrom SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS.6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, andSEQ ID NOS. 8816-8819. In preferred embodiments, the at least one targetgene is an endogenous gene of a plant, and expression of the endogenousgene is suppressed in cells of the plant where native expression of theendogenous miRNA occurs, and thus expression of the recombinant DNAconstruct in the cells results in expression of the endogenous gene inthe cells.

The DNA element for suppressing expression includes at least one of:

-   -   (a) DNA that includes at least one anti-sense DNA segment that        is anti-sense to at least one segment of the target gene;    -   (b) DNA that includes multiple copies of at least one anti-sense        DNA segment that is anti-sense to at least one segment of the        target gene;    -   (c) DNA that includes at least one sense DNA segment that is at        least one segment of the target gene;    -   (d) DNA that includes multiple copies of at least one sense DNA        segment that is at least one segment of the target gene;    -   (e) DNA that transcribes to RNA for suppressing the target gene        by forming double-stranded RNA and includes at least one        anti-sense DNA segment that is anti-sense to at least one        segment of the target gene and at least one sense DNA segment        that is at least one segment of the target gene;    -   (f) DNA that transcribes to RNA for suppressing the target gene        by forming a single double-stranded RNA and includes multiple        serial anti-sense DNA segments that are anti-sense to at least        one segment of the target gene and multiple serial sense DNA        segments that are at least one segment of the target gene;    -   (g) DNA that transcribes to RNA for suppressing the target gene        by forming multiple double strands of RNA and includes multiple        anti-sense DNA segments that are anti-sense to at least one        segment of the target gene and multiple sense DNA segments that        are at least one segment of the target gene, and wherein the        multiple anti-sense DNA segments and the multiple sense DNA        segments are arranged in a series of inverted repeats;    -   (h) DNA that includes nucleotides derived from a plant miRNA;    -   (i) DNA that includes nucleotides of a siRNA;    -   (j) DNA that transcribes to an RNA aptamer capable of binding to        a ligand; and    -   (k) DNA that transcribes to an RNA aptamer capable of binding to        a ligand, and DNA that transcribes to regulatory RNA capable of        regulating expression of the target gene, wherein the regulation        is dependent on the conformation of the regulatory RNA, and the        conformation of the regulatory RNA is allosterically affected by        the binding state of the RNA aptamer.

DNA elements for suppressing expression are described further in Example3 and depicted in FIGS. 2 and 3.

In some embodiments, the recombinant DNA construct includes DNA designedto be transcribed to single-stranded RNA or to at least partiallydouble-stranded RNA (such as in a “kissing stem-loop” arrangement), orto an RNA that assumes a secondary structure or three-dimensionalconfiguration (e.g., a large loop of antisense sequence of the targetgene or an aptamer) that confers on the transcript an additional desiredcharacteristic, such as increased stability, increased half-life invivo, or cell or tissue specificity. In one example, the spacer istranscribed to a stabilizing loop that links the first and second seriesof contiguous RNA segments (see, for example, Di Giusto and King (2004)J. Biol. Chem., 279:46483-46489). In another example, the recombinantDNA construct includes DNA that transcribes to RNA including an RNAaptamer (e.g., an aptamer that binds to a cell-specific ligand) thatallows cell- or tissue-specific targetting of the recombinant RNAduplex.

The recombinant DNA construct is made by commonly used techniques, suchas those described under the heading “Making and Using Recombinant DNAConstructs” and illustrated in the working Examples. The recombinant DNAconstruct is particularly useful for making non-natural transgenic plantcells, non-natural transgenic plants, and transgenic seeds as discussedbelow under “Transgenic Plant Cells and Transgenic Plants”.

The effects of a miRNA on its target gene can be controlled byalternative methods described in detail below under “MicroRNA DecoySequences”.

Target Genes

The recombinant DNA construct of this invention can be designed tosuppress any target gene or genes. The target gene can be translatable(coding) sequence, or can be non-coding sequence (such as non-codingregulatory sequence), or both, and can include at least one geneselected from the group consisting of a eukaryotic target gene, anon-eukaryotic target gene, a microRNA precursor DNA sequence, and amicroRNA promoter. The target gene can be native (endogenous) to thecell (e.g., a cell of a plant or animal) in which the recombinant DNAconstruct is transcribed, or can be native to a pest or pathogen of theplant or animal in which the recombinant DNA construct is transcribed.The target gene can be an exogenous gene, such as a transgene in aplant. A target gene can be a native gene targeted for suppression, withor without concurrent expression of an exogenous transgene, for example,by including a gene expression element in the recombinant DNA construct,or in a separate recombinant DNA construct. For example, it can bedesirable to replace a native gene with an exogenous transgenehomologue.

The target gene can include a single gene or part of a single gene thatis targeted for suppression, or can include, for example, multipleconsecutive segments of a target gene, multiple non-consecutive segmentsof a target gene, multiple alleles of a target gene, or multiple targetgenes from one or more species. A target gene can include any sequencefrom any species (including, but not limited to, non-eukaryotes such asbacteria, and viruses; fungi; plants, including monocots and dicots,such as crop plants, ornamental plants, and non-domesticated or wildplants; invertebrates such as arthropods, annelids, nematodes, andmolluscs; and vertebrates such as amphibians, fish, birds, domestic orwild mammals, and even humans.

In one embodiment, the target gene is exogenous to the plant in whichthe recombinant DNA construct is to be transcribed, but endogenous to apest or pathogen (e.g., viruses, bacteria, fungi, oomycetes, andinvertebrates such as insects, nematodes, and molluscs) of the plant.The target gene can include multiple target genes, or multiple segmentsof one or more genes. In one preferred embodiment, the target gene orgenes is a gene or genes of an invertebrate pest or pathogen of theplant. These embodiments are particularly useful in providingnon-natural transgenic plants having resistance to one or more plantpests or pathogens, for example, resistance to a nematode such assoybean cyst nematode or root knot nematode or to a pest insect.

The target gene can be translatable (coding) sequence, or can benon-coding sequence (such as non-coding regulatory sequence), or both.Non-limiting examples of a target gene include non-translatable(non-coding) sequence, such as, but not limited to, 5′ untranslatedregions, promoters, enhancers, or other non-coding transcriptionalregions, 3′ untranslated regions, terminators, and introns. Target genesinclude genes encoding microRNAs, small interfering RNAs, RNA componentsof ribosomes or ribozymes, small nucleolar RNAs, and other non-codingRNAs (see, for example, non-coding RNA sequences provided publicly atrfam.wustl.edu; Erdmann et al. (2001) Nucleic Acids Res., 29:189-193;Gottesman (2005) Trends Genet., 21:399-404; Griffiths-Jones et al.(2005) Nucleic Acids Res., 33:121-124). One specific example of a targetgene includes a microRNA recognition site (that is, the site on an RNAstrand to which a mature miRNA binds and induces cleavage). Anotherspecific example of a target gene includes a microRNA precursor sequencenative to a pest or pathogen of the non-natural transgenic plant, thatis, the primary transcript encoding a microRNA, or the RNA intermediatesprocessed from this primary transcript (e.g., a nuclear-limitedpri-miRNA or a pre-miRNA which can be exported from the nucleus into thecytoplasm). See, for example, Lee et al. (2002) EMBO Journal,21:4663-4670; Reinhart et al. (2002) Genes & Dev., 16:161611626; Lund etal. (2004) Science, 303:95-98; and Millar and Waterhouse (2005) Funct.Integr. Genomics, 5:129-135. Target genes can also include translatable(coding) sequence for genes encoding transcription factors and genesencoding enzymes involved in the biosynthesis or catabolism of moleculesof interest (such as, but not limited to, amino acids, fatty acids andother lipids, sugars and other carbohydrates, biological polymers, andsecondary metabolites including alkaloids, terpenoids, polyketides,non-ribosomal peptides, and secondary metabolites of mixed biosyntheticorigin).

In many preferred embodiments, the target gene is an essential gene of aplant pest or pathogen. Essential genes include genes that are requiredfor development of the pest or pathogen to a fertile reproductive adult.Essential genes include genes that, when silenced or suppressed, resultin the death of the organism (as an adult or at any developmental stage,including gametes) or in the organism's inability to successfullyreproduce (e.g., sterility in a male or female parent or lethality tothe zygote, embryo, or larva). A description of nematode essential genesis found, e.g., in Kemphues, K. “Essential Genes” (Dec. 24, 2005),WormBook, ed. The C. elegans Research Community, WormBook,doi/10.1895/wormbook.1.57.1, available on line at www.wormbook.org.Non-limiting examples of nematode essential genes include major spermprotein, RNA polymerase II, and chitin synthase (see, e.g., U.S. PatentApplication Publication US20040098761 A1); additional soybean cystnematode essential genes are provided in U.S. patent application Ser.No. 11/360,355, filed 23 Feb. 2006, incorporated by reference herein. Adescription of insect genes is publicly available at the Drosophilagenome database (available on line at flybase.bio.indiana.edu/). Themajority of predicted Drosophila genes have been analyzed for functionby a cell culture-based RNA interference screen, resulting in 438essential genes being identified; see Boutros et al. (2004) Science,303:832-835, and supporting material available on line atwww.sciencemag.org/cgi/content/full/303/5659/832/DC1. A description ofbacterial and fungal essential genes is provided in the Database ofEssential Genes (“DEG”, available on line at tubic.tju.edu.cn/deg/); seeZhang et al. (2004) Nucleic Acids Res., 32:D271-D272.

Plant pest invertebrates include, but are not limited to, pestnematodes, pest molluscs (slugs and snails), and pest insects. Plantpathogens of interest include fungi, oomycetes, bacteria (e.g., thebacteria that cause leaf spotting, fireblight, crown gall, and bacterialwilt), mollicutes, and viruses (e.g., the viruses that cause mosaics,vein banding, flecking, spotting, or abnormal growth). See also G. N.Agrios, “Plant Pathology” (Fourth Edition), Academic Press, San Diego,1997, 635 pp., for descriptions of fungi, bacteria, mollicutes(including mycoplasmas and spiroplasmas), viruses, nematodes, parasitichigher plants, and flagellate protozoans, all of which are plant pestsor pathogens of interest. See also the continually updated compilationof plant pests and pathogens and the diseases caused by such on theAmerican Phytopathological Society's “Common Names of Plant Diseases”,compiled by the Committee on Standardization of Common Names for PlantDiseases of The American Phytopathological Society, 1978-2005, availableonline at www.apsnet.org/online/common/top.asp.

Non-limiting examples of fungal plant pathogens of particular interestinclude, e.g., the fungi that cause powdery mildew, rust, leaf spot andblight, damping-off, root rot, crown rot, cotton boll rot, stem canker,twig canker, vascular wilt, smut, or mold, including, but not limitedto, Fusarium spp., Phakospora spp., Rhizoctonia spp., Aspergillus spp.,Gibberella spp., Pyricularia spp., and Alternaria spp. Specific examplesof fungal plant pathogens include Phakospora pachirhizi (Asian soybeanrust), Puccinia sorghi (corn common rust), Puccinia polysora (cornSouthern rust), Fusarium oxysporum and other Fusarium spp., Alternariaspp., Penicillium spp., Rhizoctonia solani, Exserohilum turcicum(Northern corn leaf blight), Bipolaris maydis (Southern corn leafblight), Ustilago maydis (corn smut), Fusarium graminearum (Gibberellazeae), Fusarium verticilliodes (Gibberella moniliformis), F.proliferatum (G. fujikuroi var. intermedia), F. subglutinans (G.subglutinans), Diplodia maydis, Sporisorium holci-sorghi, Colletotrichumgraminicola, Setosphaeria turcica, Aureobasidium zeae, Sclerotiniasclerotiorum, and the numerous fungal species provided in Tables 4 and 5of U.S. Pat. No. 6,194,636, which is incorporated in its entirety byreference herein. Non-limiting examples of plant pathogens includepathogens previously classified as fungi but more recently classified asoomycetes. Specific examples of oomycete plant pathogens of particularinterest include members of the genus Pythium (e.g., Pythiumaphanidermatum) and Phytophthora (e.g., Phytophthora infestans,Phytophthora sojae,) and organisms that cause downy mildew (e.g.,Peronospora farinosa).

Non-limiting examples of bacterial pathogens include the mycoplasmasthat cause yellows disease and spiroplasmas such as Spiroplasmakunkelii, which causes corn stunt, eubacteria such as Pseudomonasavenae, Pseudomonas andropogonis, Erwinia stewartii, Pseudomonassyringae pv. syringae, Xylella fastidiosa, and the numerous bacterialspecies listed in Table 3 of U.S. Pat. No. 6,194,636, which isincorporated in its entirety by reference herein.

Non-limiting examples of viral plant pathogens of particular interestinclude maize dwarf mosaic virus (MDMV), sugarcane mosaic virus (SCMV,formerly MDMV strain B), wheat streak mosaic virus (WSMV), maizechlorotic dwarf virus (MCDV), barley yellow dwarf virus (BYDV), bananabunchy top virus (BBTV), and the numerous viruses listed in Table 2 ofU.S. Pat. No. 6,194,636, which is incorporated in its entirety byreference herein.

Non-limiting examples of invertebrate pests include cyst nematodesHeterodera spp. especially soybean cyst nematode Heterodera glycines,root knot nematodes Meloidogyne spp., lance nematodes Hoplolaimus spp.,stunt nematodes Tylenchorhynchus spp., spiral nematodes Helicotylenchusspp., lesion nematodes Pratylenchus spp., ring nematodes Criconema spp.,foliar nematodes Aphelenchus spp. or Aphelenchoides spp., cornrootworms, Lygus spp., aphids and similar sap-sucking insects such asphylloxera (Daktulosphaira vitifoliae), corn borers, cutworms,armyworms, leafhoppers, Japanese beetles, grasshoppers, and other pestcoleopterans, dipterans, and lepidopterans. Specific examples ofinvertebrate pests include pests capable of infesting the root systemsof crop plants, e.g., northern corn rootworm (Diabrotica barberi),southern corn rootworm (Diabrotica undecimpunctata), Western cornrootworm (Diabrotica virgifera), corn root aphid (Anuraphismaidiradicis), black cutworm (Agrotis ipsilon), glassy cutworm (Crymodesdevastator), dingy cutworm (Feltia ducens), claybacked cutworm (Agrotisgladiaria), wireworm (Melanotus spp., Aeolus mellillus), wheat wireworm(Aeolus mancus), sand wireworm (Horistonotus uhlerii), maize billbug(Sphenophorus maidis), timothy billbug (Sphenophorus zeae), bluegrassbillbug (Sphenophorus parvulus), southern corn billbug (Sphenophoruscallosus), white grubs (Phyllophaga spp.), seedcorn maggot (Deliaplatura), grape colaspis (Colaspis brunnea), seedcorn beetle(Stenolophus lecontei), and slender seedcorn beetle (Cliviniaimpressifrons), as well as the parasitic nematodes listed in Table 6 ofU.S. Pat. No. 6,194,636, which is incorporated in its entirety byreference herein.

Invertebrate pests of particular interest, especially in but not limitedto southern hemisphere regions (including South and Central America)include aphids, corn rootworms, spodoptera, noctuideae, potato beetle,Lygus spp., any hemipteran, homopteran, or heteropteran, anylepidopteran, any coleopteran, nematodes, cutworms, earworms, armyworms,borers, leaf rollers, and others. Arthropod pests specificallyencompassed by this invention include various cutworm species includingcutworm (Agrotis repleta), black cutworm (Agrotis ipsilon), cutworm(Anicla ignicans), granulate cutworm (Feltia subterranea),“gusanoaspero” (Agrotis malefida); Mediterranean flour moth (Anagastakuehniella), square-necked grain beetle (Cathartus quadricollis), fleabeetle (Chaetocnema spp), rice moth (Corcyra cephalonica), corn rootwormor “vaquita de San Antonio” (Diabotica speciosa), sugarcane borer(Diatraea saccharalis), lesser cornstalk borer (Elasmopalpuslignosellus), brown stink bug (Euschistus spp.), corn earworm(Helicoverpa zea), flat grain beetle (Laemophloeus minutus), grasslooper moth (Mocis latipes), sawtoothed grain beetle (Oryzaephilussurinamensis), meal moth (Pyralis farinalis), Indian meal moth (Plodiainterpunctella), corn leaf aphid (Rhopalosiphum maidis), brown burrowingbug or “chinche subterranea” (Scaptocoris castanea), greenbug(Schizaphis graminum), grain weevil (Sitophilus zeamais), Angoumoisgrain moth (Sitotroga cerealella), fall armyworm (Spodopterafrugiperda), cadelle beetle (Tenebroides mauritanicus), two-spottedspider mite (Tetranychus urticae), red flour beetle (Triboleumcastaneum), cotton leafworm (Alabama argillacea), boll weevil(Anthonomus grandis), cotton aphid (Aphis gossypii), sweet potatowhitefly (Bemisia tabaci), various thrips species (Frankliniella spp.),cotton earworm (Helicoverpa zea), “oruga bolillera” (e.g., Helicoverpageletopoeon), tobacco budworm (Heliothis virescens), stinkbug (Nezaraviridula), pink bollworm (Pectinophora gossypiella), beet armyworm(Spodoptera exigua), spider mites (Tetranychus spp.), onion thrips(Thrips tabaci), greenhouse whitefly (Trialeurodes vaporarium),velvetbean caterpillar (Anticarsia gemmatalis), spotted maize beetle or“astilo moteado” (Astylus atromaculatus),“oruga de la alfalfa” (Coliaslesbia),“chinche macron” or “chinche de los cuernos” (Dichelopsfurcatus),“alquiche chico” (Edessa miditabunda), blister beetles(Epicauta spp.), “barrenador del brote” (Epinotia aporema),“oruga verdedel yuyo colorado” (Loxostege bifidalis), rootknot nematodes(Meloidogyne spp.), “oruga cuarteadora” (Mocis repanda), southern greenstink bug (Nezara viridula), “chinche de la alfalfa” (Piezodorusguildinii), green cloverworm (Plathypena scabra), soybean looper(Pseudoplusia includens), looper moth “isoca medidora del girasol”(Rachiplusia nu), yellow woolybear (Spilosoma virginica), yellowstripedarmyworm (Spodoptera ornithogalli), various root weevils (familyCurculionidae), various wireworms (family Elateridae), and various whitegrubs (family Scarabaeidae). Nematode pests specifically encompassed bythis invention include nematode pests of maize (Belonolaimus spp.,Trichodorus spp., Longidorus spp., Dolichodorus spp., Anguina spp.,Pratylenchus spp., Meloidogyne spp., Heterodera spp.), soybean(Heterodera glycines, Meloidogyne spp., Belonolaimus spp.), bananas(Radopholus similis, Meloidogyne spp., Helicotylenchus spp.), sugarcane(Heterodera sacchari, Pratylenchus spp., Meloidogyne spp.), oranges(Tylenchulus spp., Radopholus spp., Belonolaimus spp., Pratylenchusspp., Xiphinema spp.), coffee (Meloidogyne spp., Pratylenchus spp.),coconut palm (Bursaphelenchus spp.), tomatoes (Meloidogyne spp.,Belonolaimus spp., Nacobbus spp.), grapes (Meloidogyne spp., Xiphinemaspp., Tylenchulus spp., Criconemella spp.), lemon and lime (Tylenchulusspp., Radopholus spp., Belonolaimus spp., Pratylenchus spp., Xiphinemaspp.), cacao (Meloidogyne spp., Rotylenchulus reniformis), pineapple(Meloidogyne spp., Pratylenchus spp., Rotylenchulus reniformis), papaya(Meloidogyne spp., Rotylenchulus reniformis), grapefruit (Tylenchulusspp., Radopholus spp. Belonolaimus spp., Pratylenchus spp., Xiphinemaspp., and broad beans (Meloidogyne spp.).

Target genes from pests can include invertebrate genes for major spermprotein, alpha tubulin, beta tubulin, vacuolar ATPase,glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase II, chitinsynthase, cytochromes, miRNAs, miRNA precursor molecules, miRNApromoters, as well as other genes such as those disclosed in U.S. PatentApplication Publication 2006/0021087 A1, PCT Patent ApplicationPCT/US05/11816, and in Table II of U.S. Patent Application Publication2004/0098761 A1, which are incorporated by reference herein. Targetgenes from pathogens can include genes for viral translation initiationfactors, viral replicases, miRNAs, miRNA precursor molecules, fungaltubulin, fungal vacuolar ATPase, fungal chitin synthase, fungal MAPkinases, fungal Pacl Tyr/Thr phosphatase, enzymes involved in nutrienttransport (e.g., amino acid transporters or sugar transporters), enzymesinvolved in fungal cell wall biosynthesis, cutinases, melaninbiosynthetic enzymes, polygalacturonases, pectinases, pectin lyases,cellulases, proteases, genes that interact with plant avirulence genes,and other genes involved in invasion and replication of the pathogen inthe infected plant. Thus, a target gene need not be endogenous to theplant in which the recombinant DNA construct is transcribed. Arecombinant DNA construct of this invention can be transcribed in aplant and used to suppress a gene of a pathogen or pest that may infestthe plant.

Specific, non-limiting examples of suitable target genes also includeamino acid catabolic genes (such as, but not limited to, the maizeLKR/SDH gene encoding lysine-ketoglutarate reductase (LKR) andsaccharopine dehydrogenase (SDH), and its homologues), maize zein genes,genes involved in fatty acid synthesis (e.g., plant microsomal fattyacid desaturases and plant acyl-ACP thioesterases, such as, but notlimited to, those disclosed in U.S. Pat. Nos. 6,426,448, 6,372,965, and6,872,872), genes involved in multi-step biosynthesis pathways, where itmay be of interest to regulate the level of one or more intermediates,such as genes encoding enzymes for polyhydroxyalkanoate biosynthesis(see, for example, U.S. Pat. No. 5,750,848); and genes encodingcell-cycle control proteins, such as proteins with cyclin-dependentkinase (CDK) inhibitor-like activity (see, for example, genes disclosedin International Patent Application Publication Number WO 05007829A2).Target genes can include genes encoding undesirable proteins (e.g.,allergens or toxins) or the enzymes for the biosynthesis of undesirablecompounds (e.g., undesirable flavor or odor components). Thus, oneembodiment of the invention is a non-natural transgenic plant or tissueof such a plant that is improved by the suppression of allergenicproteins or toxins, e.g., a peanut, soybean, or wheat kernel withdecreased allergenicity. Target genes can include genes involved infruit ripening, such as polygalacturonase. Target genes can includegenes where expression is preferably limited to a particular cell ortissue or developmental stage, or where expression is preferablytransient, that is to say, where constitutive or general suppression, orsuppression that spreads through many tissues, is not necessarilydesired. Thus, other examples of suitable target genes include genesencoding proteins that, when expressed in transgenic plants, make thetransgenic plants resistant to pests or pathogens (see, for example,genes for cholesterol oxidase as disclosed in U.S. Pat. No. 5,763,245);genes where expression is pest- or pathogen-induced; and genes which caninduce or restore fertility (see, for example, the barstar/barnase genesdescribed in U.S. Pat. No. 6,759,575); all the patents cited in thisparagraph are incorporated by reference in their entirety herein.

The recombinant DNA construct can be designed to be more specificallysuppress the target gene, for example, by designing the recombinant DNAconstruct to encode a mature miRNA to include regions substantiallynon-identical (or non-complementary) to a non-target gene sequence.Non-target genes can include any gene not intended to be silenced orsuppressed, either in a plant containing the recombinant DNA constructor in organisms that may come into contact with the recombinant DNAconstruct. A non-target gene sequence can include any sequence from anyspecies (including, but not limited to, non-eukaryotes such as bacteria,and viruses; fungi; plants, including monocots and dicots, such as cropplants, ornamental plants, and non-domesticated or wild plants;invertebrates such as arthropods, annelids, nematodes, and molluscs; andvertebrates such as amphibians, fish, birds, domestic or wild mammals,and even humans).

In one embodiment, the target gene is a gene endogenous to a givenspecies, such as a given plant (such as, but not limited to,agriculturally or commercially important plants, including monocots anddicots), and the non-target gene can be, e.g., a gene of a non-targetspecies, such as another plant species or a gene of a virus, fungus,bacterium, invertebrate, or vertebrate, even a human. One non-limitingexample is where the recombinant DNA construct is designed to suppress atarget gene that is a gene endogenous to a single species (e.g., Westerncorn rootworm, Diabrotica virgifera virgifera LeConte) but to notsuppress a non-target gene such as genes from related, even closelyrelated, species (e.g., Northern corn rootworm, Diabrotica barberi Smithand Lawrence, or Southern corn rootworm, Diabrotica undecimpunctata).

In other embodiments (e.g., where it is desirable to suppress a targetgene across multiple species), it may be desirable to design therecombinant DNA construct to suppress a target gene sequence common tothe multiple species in which the target gene is to be silenced. Thus,an RNA duplex can be selected to be specific for one taxon (for example,specific to a genus, family, or even a larger taxon such as a phylum,e.g., arthropoda) but not for other taxa (e.g., plants or vertebrates ormammals). In one non-limiting example of this embodiment, a recombinantDNA construct for gene silencing can be selected so as to targetpathogenic fungi (e.g., a Fusarium spp.) but not target any genesequence from beneficial fungi.

In another non-limiting example of this embodiment, a recombinant DNAconstruct for gene silencing in corn rootworm can be selected to bespecific to all members of the genus Diabrotica. In a further example ofthis embodiment, such a Diabrotica-targeted recombinant DNA constructcan be selected so as to not target any gene sequence from beneficialcoleopterans (for example, predatory coccinellid beetles, commonly knownas ladybugs or ladybirds) or other beneficial insect species.

The required degree of specificity of a recombinant DNA construct ofthis invention for silencing a target gene depends on various factors.Factors can include the size and nucleic acid sequence of a maturemicroRNA encoded by the recombinant DNA construct, and the relativeimportance of decreasing such a mature miRNA's potential to suppressnon-target genes. In a non-limiting example, where such a mature miRNAis expected to be 21 base pairs in size, one particularly preferredembodiment includes DNA encoding a mature miRNA for silencing a targetgene wherein the mature miRNA includes sequence that is substantiallynon-identical to a non-target gene sequence, such as fewer than 18, orfewer than 17, or fewer than 16, or fewer than 15 matches out of 21contiguous nucleotides of a non-target gene sequence.

In some embodiments, it may be desirable to design the recombinant DNAconstruct for silencing a target gene to include regions predicted tonot generate undesirable polypeptides, for example, by screening therecombinant DNA construct for sequences that may encode knownundesirable polypeptides or close homologues of these. Undesirablepolypeptides include, but are not limited to, polypeptides homologous toknown allergenic polypeptides and polypeptides homologous to knownpolypeptide toxins. Publicly available sequences encoding suchundesirable potentially allergenic peptides are available, for example,the Food Allergy Research and Resource Program (FARRP) allergen database(available at allergenonline.com) or the Biotechnology Information forFood Safety Databases (available at www.iit.edu/˜sgendel/fa.htm) (seealso, for example, Gendel (1998) Adv. Food Nutr. Res., 42:63-92).Undesirable sequences can also include, for example, those polypeptidesequences annotated as known toxins or as potential or known allergensand contained in publicly available databases such as GenBank, EMBL,SwissProt, and others, which are searchable by the Entrez system(www.ncbi.nih.gov/Entrez). Non-limiting examples of undesirable,potentially allergenic peptide sequences include glycinin from soybean,oleosin and agglutinin from peanut, glutenins from wheat, casein,lactalbumin, and lactoglobulin from bovine milk, and tropomyosin fromvarious shellfish (allergenonline.com). Non-limiting examples ofundesirable, potentially toxic peptides include tetanus toxin tetA fromClostridium tetani, diarrheal toxins from Staphylococcus aureus, andvenoms such as conotoxins from Conus spp. and neurotoxins fromarthropods and reptiles (www.ncbi.nih.gov/Entrez).

In one non-limiting example, the recombinant DNA construct is screenedto eliminate those transcribable sequences encoding polypeptides withperfect homology to a known allergen or toxin over 8 contiguous aminoacids, or with at least 35% identity over at least 80 amino acids; suchscreens can be performed on any and all possible reading frames in bothdirections, on potential open reading frames that begin with AUG (ATG inthe corresponding DNA), or on all possible reading frames, regardless ofwhether they start with an AUG (or ATG) or not. When a “hit” or match ismade, that is, when a sequence that encodes a potential polypeptide withperfect homology to a known allergen or toxin over 8 contiguous aminoacids (or at least about 35% identity over at least about 80 aminoacids), is identified, the nucleic acid sequences corresponding to thehit can be avoided, eliminated, or modified when selecting sequences tobe used in an RNA for silencing a target gene. In one embodiment therecombinant DNA construct is designed so no potential open reading framethat begins with AUG (ATG in the corresponding DNA) is included.

Avoiding, elimination of, or modification of, an undesired sequence canbe achieved by any of a number of methods known to those skilled in theart. In some cases, the result can be novel sequences that are believedto not exist naturally. For example, avoiding certain sequences can beaccomplished by joining together “clean” sequences into novel chimericsequences to be used in the RHA duplex.

Applicants recognize that in some microRNA-mediated gene silencing, itis possible for imperfectly matching miRNA sequences to be effective atgene silencing. For example, it has been shown that mismatches near thecenter of a miRNA complementary site has stronger effects on the miRNA'sgene silencing than do more distally located mismatches. See, forexample, FIG. 4 in Mallory et al. (2004) EMBO J., 23:3356-3364. Inanother example, it has been reported that, both the position of amismatched base pair and the identity of the nucleotides forming themismatch influence the ability of a given siRNA to silence a targetgene, and that adenine-cytosine mismatches, in addition to the G:Uwobble base pair, were well tolerated (see Du et al. (2005) NucleicAcids Res., 33:1671-1677). Thus, a given strand of the recombinant DNAconstruct need not always have 100% sequence identity with the intendedtarget gene, but generally would preferably have substantial sequenceidentity with the intended target gene, such as about 95%, about 90%,about 85%, or about 80% sequence identity with the intended target gene.Described in terms of complementarity, one strand of the recombinant DNAconstruct is preferably designed to have substantial complementarity tothe intended target (e.g., a target messenger RNA or target non-codingRNA), such as about 95%, about 90%, about 85%, or about 80%complementarity to the intended target. In a non-limiting example, inthe case of a recombinant DNA construct encoding a mature miRNA of 21nucleotides, the encoded mature miRNA is designed to be is substantiallybut not perfectly complementary to 21 contiguous nucleotides of a targetRNA; preferably the nucleotide at position 21 is unpaired with thecorresponding position in the target RNA to prevent transitivity.

One skilled in the art would be capable of judging the importance givento screening for regions predicted to be more highly specific to thetarget gene or predicted to not generate undesirable polypeptides,relative to the importance given to other criteria, such as, but notlimited to, the percent sequence identity with the intended target geneor the predicted gene silencing efficiency of a given sequence. Forexample, a recombinant DNA construct of this invention that encodes amature miRNA may be designed to be active across several species, andtherefore one skilled in the art can determine that it is more importantto include in the recombinant DNA construct DNA encoding a mature miRNAthat is specific to the several species of interest, but less importantto screen for regions predicted to have higher gene silencing efficiencyor for regions predicted to generate undesirable polypeptides.

Promoters

Generally, the recombinant DNA construct of this invention includes apromoter, functional in a plant cell, and operably linked to thetranscribable DNA element. In various embodiments, the promoter isselected from the group consisting of a constitutive promoter, aspatially specific promoter, a temporally specific promoter, adevelopmentally specific promoter, and an inducible promoter.

Non-constitutive promoters suitable for use with the recombinant DNAconstructs of the invention include spatially specific promoters,temporally specific promoters, and inducible promoters. Spatiallyspecific promoters can include organelle-, cell-, tissue-, ororgan-specific promoters (e.g., a plastid-specific, a root-specific, apollen-specific, or a seed-specific promoter for suppressing expressionof the first target RNA in plastids, roots, pollen, or seeds,respectively). In many cases a seed-specific, embryo-specific,aleurone-specific, or endosperm-specific promoter is especially useful.Temporally specific promoters can include promoters that tend to promoteexpression during certain developmental stages in a plant's growthcycle, or during different times of day or night, or at differentseasons in a year. Inducible promoters include promoters induced bychemicals or by environmental conditions such as, but not limited to,biotic or abiotic stress (e.g., water deficit or drought, heat, cold,high or low nutrient or salt levels, high or low light levels, or pestor pathogen infection). An expression-specific promoter can also includepromoters that are generally constitutively expressed but at differingdegrees or “strengths” of expression, including promoters commonlyregarded as “strong promoters” or as “weak promoters”.

Promoters of particular interest include the following non-limitingexamples: an opaline synthase promoter isolated from T-DNA ofAgrobacterium; a cauliflower mosaic virus 35S promoter; enhancedpromoter elements or chimeric promoter elements such as an enhancedcauliflower mosaic virus (CaMV) 35S promoter linked to an enhancerelement (an intron from heat shock protein 70 of Zea mays); rootspecific promoters such as those disclosed in U.S. Pat. Nos. 5,837,848;6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in U.S.Pat. No. 6,433,252; a promoter for a plant nuclear gene encoding aplastid-localized aldolase disclosed in U.S. Patent ApplicationPublication 2004/0216189; cold-inducible promoters disclosed in U.S.Pat. No. 6,084,089; salt-inducible promoters disclosed in U.S. Pat. No.6,140,078; light-inducible promoters disclosed in U.S. Pat. No.6,294,714; pathogen-inducible promoters disclosed in U.S. Pat. No.6,252,138; and water deficit-inducible promoters disclosed in U.S.Patent Application Publication 2004/0123347 A1. All of theabove-described patents and patent publications disclosing promoters andtheir use, especially in recombinant DNA constructs functional in plantsare incorporated herein by reference.

The promoter element can include nucleic acid sequences that are notnaturally occurring promoters or promoter elements or homologues thereofbut that can regulate expression of a gene. Examples of such “geneindependent” regulatory sequences include naturally occurring orartificially designed RNA sequences that include a ligand-binding regionor aptamer and a regulatory region (which can be cis-acting). See, forexample, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer andSmolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004)Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005)Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature,419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal andBreaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such“riboregulators” can be selected or designed for specific spatial ortemporal specificity, for example, to regulate translation of theexogenous gene only in the presence (or absence) of a givenconcentration of the appropriate ligand.

Making and Using Recombinant DNA Constructs

The recombinant DNA constructs of this invention are made by any methodsuitable to the intended application, taking into account, for example,the type of expression desired and convenience of use in the plant inwhich the construct is to be transcribed. General methods for making andusing DNA constructs and vectors are well known in the art and describedin detail in, for example, handbooks and laboratory manuals includingSambrook and Russell, “Molecular Cloning: A Laboratory Manual” (thirdedition), Cold Spring Harbor Laboratory Press, NY, 2001. An example ofuseful technology for building DNA constructs and vectors fortransformation is disclosed in U.S. Patent Application Publication2004/0115642 A1, incorporated herein by reference. DNA constructs canalso be built using the GATEWAYTm cloning technology (available fromInvitrogen Life Technologies, Carlsbad, Calif.), which uses thesite-specific recombinase LR cloning reaction of the Integrase/attsystem from bacteriophage lambda vector construction, instead ofrestriction endonucleases and ligases. The LR cloning reaction isdisclosed in U.S. Pat. Nos. 5,888,732 and 6,277,608, and in U.S. PatentApplication Publications 2001/283529, 2001/282319 and 2002/0007051, allof which are incorporated herein by reference. The GATEWAY™ CloningTechnology Instruction Manual, which is also supplied by Invitrogen,provides concise directions for routine cloning of any desired DNA intoa vector comprising operable plant expression elements. Anotheralternative vector fabrication method employs ligation-independentcloning as disclosed by Aslandis et al. (1990) Nucleic Acids Res.,18:6069-6074 and Rashtchian et al. (1992) Biochem., 206:91-97, where aDNA fragment with single-stranded 5′ and 3′ ends is ligated into adesired vector which can then be amplified in vivo.

In certain embodiments, the DNA sequence of the recombinant DNAconstruct includes sequence that has been codon-optimized for the plantin which the recombinant DNA construct is to be expressed. For example,a recombinant DNA construct to be expressed in a plant can have all orparts of its sequence (e.g., the first gene suppression element or thegene expression element) codon-optimized for expression in a plant bymethods known in the art. See, e.g., U.S. Pat. No. 5,500,365,incorporated by reference, for a description of codon-optimization forplants; see also De Amicis and Marchetti (2000) Nucleic Acid Res.,28:3339-3346.

Transgenic Plant Cells and Plants

Another aspect of this invention provides a non-natural transgenic plantcell including any of the recombinant DNA constructs of this invention,as described above under the heading “Recombinant DNA Constructs”.Further provided is a non-natural transgenic plant containing thenon-natural transgenic plant cell of this invention. The non-naturaltransgenic plant of this invention includes plants of any developmentalstage, and includes a regenerated plant prepared from the transgenicplant cells disclosed herein, or a progeny plant (which can be an inbredor hybrid progeny plant) of the regenerated plant, or seed of such atransgenic plant. Also provided and claimed is a transgenic seed havingin its genome any of the recombinant DNA constructs provided by thisinvention. The non-natural transgenic plant cells, non-naturaltransgenic plants, and transgenic seeds of this invention are made bymethods well-known in the art, as described below under the heading“Making and Using Non-natural Transgenic Plant Cells and Non-naturalTransgenic Plants”.

The non-natural transgenic plant cell can include an isolated plant cell(e.g., individual plant cells or cells grown in or on an artificialculture medium), or can include a plant cell in undifferentiated tissue(e.g., callus or any aggregation of plant cells). The non-naturaltransgenic plant cell can include a plant cell in at least onedifferentiated tissue selected from the group consisting of leaf (e.g.,petiole and blade), root, stem (e.g., tuber, rhizome, stolon, bulb, andcorm) stalk (e.g., xylem, phloem), wood, seed, fruit (e.g., nut, grain,fleshy fruits), and flower (e.g., stamen, filament, anther, pollen,carpel, pistil, ovary, ovules).

The non-natural transgenic plant cell or non-natural transgenic plant ofthe invention can be any suitable plant cell or plant of interest. Bothtransiently transformed and stably transformed plant cells areencompassed by this invention. Stably transformed transgenic plants areparticularly preferred. In many preferred embodiments, the non-naturaltransgenic plant is a fertile transgenic plant from which seed can beharvested, and the invention further claims transgenic seed of suchtransgenic plants, wherein the seed preferably also contains therecombinant construct of this invention.

Making and Using Non-Natural Transgenic Plant Cells and Non-NaturalTransgenic Plants

Where a recombinant DNA construct of this invention is used to produce anon-natural transgenic plant cell, non-natural transgenic plant, ortransgenic seed of this invention, transformation can include any of thewell-known and demonstrated methods and compositions. Suitable methodsfor plant transformation include virtually any method by which DNA canbe introduced into a cell, such as by direct delivery of DNA (e.g., byPEG-mediated transformation of protoplasts, by electroporation, byagitation with silicon carbide fibers, and by acceleration of DNA coatedparticles), by Agrobacterium-mediated transformation, by viral or othervectors, etc. One preferred method of plant transformation ismicroprojectile bombardment, for example, as illustrated in U.S. Pat.No. 5,015,580 (soy), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No.5,538,880 (maize), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No.6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice) and U.S. Pat. No.6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize), all of which areincorporated by reference.

Another preferred method of plant transformation isAgrobacterium-mediated transformation. In one preferred embodiment, thenon-natural transgenic plant cell of this invention is obtained bytransformation by means of Agrobacterium containing a binary Ti plasmidsystem, wherein the Agrobacterium carries a first Ti plasmid and asecond, chimeric plasmid containing at least one T-DNA border of awild-type Ti plasmid, a promoter functional in the transformed plantcell and operably linked to a gene suppression construct of theinvention. See, for example, the binary system described in U.S. Pat.No. 5,159,135, incorporated by reference. Also see De Framond (1983)Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. Insuch a binary system, the smaller plasmid, containing the T-DNA borderor borders, can be conveniently constructed and manipulated in asuitable alternative host, such as E. coli, and then transferred intoAgrobacterium.

Detailed procedures for Agrobacterium-mediated transformation of plants,especially crop plants, include, for example, procedures disclosed inU.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat.Nos. 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soy); U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,981,840 (maize); U.S. Pat. No. 5,463,174(brassicas), and in U.S. Patent Application Publication 2004/0244075(maize), all of which are incorporated by reference. Similar methodshave been reported for many plant species, both dicots and monocots,including, among others, peanut (Cheng et al. (1996) Plant Cell Rep.,15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci.U.S.A., 84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37);rice (Toriyama et al. (1988) Bio/Technology, 6:10; Zhang et al. (1988)Plant Cell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology,10:667; Becker et al. (1994) Plant J., 5:299), alfalfa (Masoud et al.(1996) Transgen. Res., 5:313); and tomato (Sun et al. (2006) Plant CellPhysiol., 47:426-431). See also a description of vectors, transformationmethods, and production of transformed Arabidopsis thaliana plants wheretranscription factors are constitutively expressed by a CaMV35Spromoter, in U.S. Patent Application Publication 2003/0167537 A1,incorporated by reference. Transgenic plant cells and transgenic plantscan also be obtained by transformation with other vectors, such as, butnot limited to, viral vectors (e.g., tobacco etch potyvirus (TEV),barley stripe mosaic virus (BSMV), and the viruses referenced inEdwardson and Christie, “The Potyvirus Group: Monograph No. 16, 1991,Agric. Exp. Station, Univ. of Florida), plasmids, cosmids, YACs (yeastartificial chromosomes), BACs (bacterial artificial chromosomes) or anyother suitable cloning vector, when used with an appropriatetransformation protocol, e.g., bacterial infection (e.g., withAgrobacterium as described above), binary bacterial artificialchromosome constructs, direct delivery of DNA (e.g., via PEG-mediatedtransformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, andmicroprojectile bombardment). It would be clear to one of ordinary skillin the art that various transformation methodologies can be used andmodified for production of stable transgenic plants from any number ofplant species of interest.

Transformation methods to provide transgenic plant cells and transgenicplants containing stably integrated recombinant DNA are preferablypracticed in tissue culture on media and in a controlled environment.“Media” refers to the numerous nutrient mixtures that are used to growcells in vitro, that is, outside of the intact living organism.Recipient cell targets include, but are not limited to, meristem cells,callus, immature embryos or parts of embryos, and gametic cells such asmicrospores, pollen, sperm, and egg cells. Any cell from which a fertileplant can be regenerated is contemplated as a useful recipient cell forpractice of the invention. Callus can be initiated from various tissuesources, including, but not limited to, immature embryos or parts ofembryos, seedling apical meristems, microspores, and the like. Thosecells which are capable of proliferating as callus can serve asrecipient cells for genetic transformation. Practical transformationmethods and materials for making non-natural transgenic plants of thisinvention (e.g., various media and recipient target cells,transformation of immature embryos, and subsequent regeneration offertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos.6,194,636 and 6,232,526 and U.S. Patent Application Publication2004/0216189, which are incorporated by reference. Transgenic plantsinclude transgenic plant tissue or parts, such as transgenic rootstockor transgenic graft or scion material, which can be used in combinationwith non-transgenic plant tissue or parts.

In general transformation practice, DNA is introduced into only a smallpercentage of target cells in any one transformation experiment. Markergenes are generally used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theantibiotics or herbicides to which a plant cell may be resistant can bea useful agent for selection. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells can be tested further to confirm stable integration of therecombinant DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin or paromomycin(nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(EPSPS). Examples of useful selective marker genes and selection agentsare illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and6,118,047, all of which are incorporated by reference. Screenablemarkers or reporters, such as markers that provide an ability tovisually identify transformants can also be employed. Non-limitingexamples of useful screenable markers include, for example, a geneexpressing a protein that produces a detectable color by acting on achromogenic substrate (e.g., beta-glucuronidase (GUS) (uidA) orluciferase (luc)) or that itself is detectable, such as greenfluorescent protein (GFP) (gfp) or an immunogenic molecule. Those ofskill in the art will recognize that many other useful markers orreporters are available for use.

Detecting or measuring the resulting change in expression of the targetgene (or concurrent expression of a gene of interest) obtained bytranscription of the recombinant construct in the non-natural transgenicplant of the invention can be achieved by any suitable methods,including protein detection methods (e.g., western blots, ELISAs, andother immunochemical methods), measurements of enzymatic activity, ornucleic acid detection methods (e.g., Southern blots, northern blots,PCR, RT-PCR, fluorescent in situ hybridization). Such methods are wellknown to those of ordinary skill in the art as evidenced by the numeroushandbooks available; see, for example, Joseph Sambrook and David W.Russell, “Molecular Cloning: A Laboratory Manual” (third edition), ColdSpring Harbor Laboratory Press, NY, 2001; Frederick M. Ausubel et al.(editors) “Short Protocols in Molecular Biology” (fifth edition), JohnWiley and Sons, 2002; John M. Walker (editor) “Protein ProtocolsHandbook” (second edition), Humana Press, 2002; and Leandro Pena(editor) “Transgenic Plants: Methods and Protocols”, Humana Press, 2004.

Other suitable methods for detecting or measuring the resulting changein expression of the target gene (or concurrent expression of a gene ofinterest) obtained by transcription of the recombinant DNA in thenon-natural transgenic plant of the invention include measurement of anyother trait that is a direct or proxy indication of expression of thetarget gene (or concurrent expression of a gene of interest) in thetransgenic plant in which the recombinant DNA is transcribed, relativeto one in which the recombinant DNA is not transcribed, e.g., gross ormicroscopic morphological traits, growth rates, yield, reproductive orrecruitment rates, resistance to pests or pathogens, or resistance tobiotic or abiotic stress (e.g., water deficit stress, salt stress,nutrient stress, heat or cold stress). Such methods can use directmeasurements of a phenotypic trait or proxy assays (e.g., in plants,these assays include plant part assays such as leaf or root assays todetermine tolerance of abiotic stress).

The recombinant DNA constructs of the invention can be stacked withother recombinant DNA for imparting additional traits (e.g., in the caseof transformed plants, traits including herbicide resistance, pestresistance, cold germination tolerance, water deficit tolerance, and thelike) for example, by expressing or suppressing other genes. Constructsfor coordinated decrease and increase of gene expression are disclosedin U.S. Patent Application Publication 2004/0126845 A1, incorporated byreference.

Seeds of transgenic, fertile plants can be harvested and used to growprogeny generations, including hybrid generations, of non-naturaltransgenic plants of this invention that include the recombinant DNAconstruct in their genome. Thus, in addition to direct transformation ofa plant with a recombinant DNA construct, non-natural transgenic plantsof the invention can be prepared by crossing a first plant having therecombinant DNA with a second plant lacking the construct. For example,the recombinant DNA can be introduced into a plant line that is amenableto transformation to produce a non-natural transgenic plant, which canbe crossed with a second plant line to introgress the recombinant DNAinto the resulting progeny. A non-natural transgenic plant of theinvention with one recombinant DNA (effecting change in expression of atarget gene) can be crossed with a plant line having other recombinantDNA that confers one or more additional trait(s) (such as, but notlimited to, herbicide resistance, pest or disease resistance,environmental stress resistance, modified nutrient content, and yieldimprovement) to produce progeny plants having recombinant DNA thatconfers both the desired target sequence expression behavior and theadditional trait(s).

Typically, in such breeding for combining traits the transgenic plantdonating the additional trait is a male line and the transgenic plantcarrying the base traits is the female line. The progeny of this crosssegregate such that some of the plant will carry the DNA for bothparental traits and some will carry DNA for one parental trait; suchplants can be identified by markers associated with parental recombinantDNA Progeny plants carrying DNA for both parental traits can be crossedback into the female parent line multiple times, e.g., usually 6 to 8generations, to produce a progeny plant with substantially the samegenotype as one original transgenic parental line but for therecombinant DNA of the other transgenic parental line.

Yet another aspect of the invention is a non-natural transgenic plantgrown from the transgenic seed of the invention. This inventioncontemplates non-natural transgenic plants grown directly fromtransgenic seed containing the recombinant DNA as well as progenygenerations of plants, including inbred or hybrid plant lines, made bycrossing a transgenic plant grown directly from transgenic seed to asecond plant not grown from the same transgenic seed.

Crossing can include, for example, the following steps:

-   -   (a) plant seeds of the first parent plant (e.g., non-transgenic        or a transgenic) and a second parent plant that is transgenic        according to the invention;    -   (b) grow the seeds of the first and second parent plants into        plants that bear flowers;    -   (c) pollinate a flower from the first parent with pollen from        the second parent; and    -   (d) harvest seeds produced on the parent plant bearing the        fertilized flower.

It is often desirable to introgress recombinant DNA into elitevarieties, e.g., by backcrossing, to transfer a specific desirable traitfrom one source to an inbred or other plant that lacks that trait. Thiscan be accomplished, for example, by first crossing a superior inbred(“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrent parent),which carries the appropriate gene(s) for the trait in question, forexample, a construct prepared in accordance with the current invention.The progeny of this cross first are selected in the resultant progenyfor the desired trait to be transferred from the non-recurrent parent“B”, and then the selected progeny are mated back to the superiorrecurrent parent “A”. After five or more backcross generations withselection for the desired trait, the progeny are hemizygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other genes. The last backcrossgeneration would be selfed to give progeny which are pure breeding forthe gene(s) being transferred, i.e., one or more transformation events.

Through a series of breeding manipulations, a selected DNA construct canbe moved from one line into an entirely different line without the needfor further recombinant manipulation. One can thus produce inbred plantswhich are true breeding for one or more DNA constructs. By crossingdifferent inbred plants, one can produce a large number of differenthybrids with different combinations of DNA constructs. In this way,plants can be produced which have the desirable agronomic propertiesfrequently associated with hybrids (“hybrid vigor”), as well as thedesirable characteristics imparted by one or more DNA constructs.

Genetic markers can be used to assist in the introgression of one ormore DNA constructs of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers can provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers can be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized. The usefulness ofmarker assisted selection in breeding non-natural transgenic plants ofthe current invention, as well as types of useful molecular markers,such as but not limited to SSRs and SNPs, are discussed in PCTApplication Publication WO 02/062129 and U.S. Patent ApplicationPublications Numbers 2002/0133852, 2003/0049612, and 2003/0005491, eachof which is incorporated by reference in their entirety.

In certain non-natural transgenic plant cells and non-natural transgenicplants of the invention, it may be desirable to concurrently express (orsuppress) a gene of interest while also regulating expression of atarget gene. Thus, in some embodiments, the non-natural transgenic plantcontains recombinant DNA further including a gene expression (orsuppression) element for expressing at least one gene of interest, andregulation of expression of a target gene is preferably effected withconcurrent expression (or suppression) of the at least one gene ofinterest in the transgenic plant.

Thus, as described herein, the non-natural transgenic plant cells ornon-natural transgenic plants of the invention can be obtained by use ofany appropriate transient or stable, integrative or non-integrativetransformation method known in the art or presently disclosed. Therecombinant DNA constructs can be transcribed in any plant cell ortissue or in a whole plant of any developmental stage. Transgenic plantscan be derived from any monocot or dicot plant, such as, but not limitedto, plants of commercial or agricultural interest, such as crop plants(especially crop plants used for human food or animal feed), wood- orpulp-producing trees, vegetable plants, fruit plants, and ornamentalplants. Non-limiting examples of plants of interest include grain cropplants (such as wheat, oat, barley, maize, rye, triticale, rice, millet,sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such asforage grasses and forage dicots including alfalfa, vetch, clover, andthe like); oilseed crop plants (such as cotton, safflower, sunflower,soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (suchas walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane,coconut, date palm, olive, sugarbeet, tea, and coffee; wood- orpulp-producing trees; vegetable crop plants such as legumes (forexample, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus,artichoke, celery, carrot, radish, the brassicas (for example, cabbages,kales, mustards, and other leafy brassicas, broccoli, cauliflower,Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example,cucumbers, melons, summer squashes, winter squashes), edible alliums(for example, onions, garlic, leeks, shallots, chives), edible membersof the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers,groundcherries), and edible members of the Chenopodiaceae (for example,beet, chard, spinach, quinoa, amaranth); fruit crop plants such asapple, pear, citrus fruits (for example, orange, lime, lemon,grapefruit, and others), stone fruits (for example, apricot, peach,plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado,and berries; and ornamental plants including ornamental floweringplants, ornamental trees and shrubs, ornamental groundcovers, andornamental grasses. Preferred dicot plants include, but are not limitedto, canola, broccoli, cabbage, carrot, cauliflower, Chinese cabbage,cucumber, dry beans, eggplant, fennel, garden beans, gourds, lettuces,melons, okra, peas, peppers, pumpkin, radishes, spinach, squash,watermelon, cotton, potato, quinoa, amaranth, buckwheat, safflower,soybean, sugarbeet, and sunflower. Preferred monocots include, but arenot limited to, wheat, oat, barley, maize (including sweet corn andother varieties), rye, triticale, rice, ornamental and forage grasses,sorghum, millet, onions, leeks, and sugarcane, more preferably maize,wheat, and rice.

The ultimate goal in plant transformation is to produce plants which areuseful to man. In this respect, non-natural transgenic plants of theinvention can be used for virtually any purpose deemed of value to thegrower or to the consumer. For example, one may wish to harvest thetransgenic plant itself, or harvest transgenic seed of the transgenicplant for planting purposes, or products can be made from the transgenicplant or its seed such as oil, starch, ethanol or other fermentationproducts, animal feed or human food, pharmaceuticals, and variousindustrial products. For example, maize is used extensively in the foodand feed industries, as well as in industrial applications. Furtherdiscussion of the uses of maize can be found, for example, in U.S. Pat.Nos. 6,194,636, 6,207,879, 6,232,526, 6,426,446, 6,429,357, 6,433,252,6,437,217, and 6,583,338, incorporated by reference, and PCTPublications WO 95/06128 and WO 02/057471. Thus, this invention alsoprovides commodity products produced from a transgenic plant cell,plant, or seed of this invention, including, but not limited to,harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or otherparts of a plant, meals, oils, extracts, fermentation or digestionproducts, crushed or whole grains or seeds of a plant, or any food ornon-food product including such commodity products produced from atransgenic plant cell, plant, or seed of this invention. The detectionof one or more of nucleic acid sequences of the recombinant DNAconstructs of this invention in one or more commodity or commodityproducts contemplated herein is de facto evidence that the commodity orcommodity product contains or is derived from a transgenic plant cell,plant, or seed of this invention.

In preferred embodiments, the non-natural transgenic plant prepared fromthe non-natural transgenic plant cell of this invention, i.e, atransgenic plant having in its genome a recombinant DNA construct ofthis invention has at least one additional altered trait, relative to aplant lacking the recombinant DNA construct, selected from the group oftraits consisting of:

-   -   (a) improved abiotic stress tolerance;    -   (b) improved biotic stress tolerance;    -   (c) modified primary metabolite composition;    -   (d) modified secondary metabolite composition;    -   (e) modified trace element, carotenoid, or vitamin composition;    -   (f) improved yield;    -   (g) improved ability to use nitrogen or other nutrients;    -   (h) modified agronomic characteristics;    -   (i) modified growth or reproductive characteristics; and    -   (j) improved harvest, storage, or processing quality.

In particularly preferred embodiments, the non-natural transgenic plantis characterized by: improved tolerance of abiotic stress (e.g.,tolerance of water deficit or drought, heat, cold, non-optimal nutrientor salt levels, non-optimal light levels) or of biotic stress (e.g.,crowding, allelopathy, or wounding); by a modified primary metabolite(e.g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate)composition; a modified secondary metabolite (e.g., alkaloids,terpenoids, polyketides, non-ribosomal peptides, and secondarymetabolites of mixed biosynthetic origin) composition; a modified traceelement (e.g., iron, zinc), carotenoid (e.g., beta-carotene, lycopene,lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin(e.g., tocopherols) composition; improved yield (e.g., improved yieldunder non-stress conditions or improved yield under biotic or abioticstress); improved ability to use nitrogen or other nutrients; modifiedagronomic characteristics (e.g., delayed ripening; delayed senescence;earlier or later maturity; improved shade tolerance; improved resistanceto root or stalk lodging; improved resistance to “green snap” of stems;modified photoperiod response); modified growth or reproductivecharacteristics (e.g., intentional dwarfing; intentional male sterility,useful, e.g., in improved hybridization procedures; improved vegetativegrowth rate; improved germination; improved male or female fertility);improved harvest, storage, or processing quality (e.g., improvedresistance to pests during storage, improved resistance to breakage,improved appeal to consumers); or any combination of these traits.

In one preferred embodiment, transgenic seed, or seed produced by thenon-natural transgenic plant, has modified primary metabolite (e.g.,fatty acid, oil, amino acid, protein, sugar, or carbohydrate)composition, a modified secondary metabolite (e.g., alkaloids,terpenoids, polyketides, non-ribosomal peptides, and secondarymetabolites of mixed biosynthetic origin) composition, a modified traceelement (e.g., iron, zinc), carotenoid (e.g., beta-carotene, lycopene,lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin(e.g., tocopherols,) composition, an improved harvest, storage, orprocessing quality, or a combination of these. For example, it can bedesirable to modify the amino acid (e.g., lysine, methionine,tryptophan, or total protein), oil (e.g., fatty acid composition ortotal oil), carbohydrate (e.g., simple sugars or starches), traceelement, carotenoid, or vitamin content of seeds of crop plants (e.g.,canola, cotton, safflower, soybean, sugarbeet, sunflower, wheat, maize,or rice), preferably in combination with improved seed harvest, storage,or processing quality, and thus provide improved seed for use in animalfeeds or human foods. In another instance, it can be desirable to changelevels of native components of the transgenic plant or seed of atransgenic plant, for example, to decrease levels of proteins with lowlevels of lysine, methionine, or tryptophan, or to increase the levelsof a desired amino acid or fatty acid, or to decrease levels of anallergenic protein or glycoprotein (e.g., peanut allergens including arah 1, wheat allergens including gliadins and glutenins, soybean allergensincluding P34 allergen, globulins, glycinins, and conglycinins) or of atoxic metabolite (e.g., cyanogenic glycosides in cassava, solanumalkaloids in members of the Solanaceae).

Methods of Gene Suppression

A further aspect of this invention provides a method of effecting genesuppression, including the steps of: (a) providing a non-naturaltransgenic plant including a regenerated plant prepared from anon-natural transgenic plant cell of this invention, or a progeny plantof the regenerated plant (as described above under the heading“Transgenic Plant Cells and Plants”); and (b) transcribing therecombinant DNA construct in the non-natural transgenic plant; whereinthe transcribing produces RNA that is capable of suppressing the atleast one target gene in the non-natural transgenic plant, and wherebythe at least one target gene is suppressed relative to its expression inthe absence of transcription of the recombinant DNA construct.

The at least one target gene is at least one gene selected from thegroup consisting of a gene native to the transgenic plant, a transgenein the transgenic plant, and a gene native to a viral, a bacterial, afungal, or an invertebrate pest or pathogen of the transgenic plant.Suitable target genes are described above under the heading “TargetGenes”. In some embodiments, the at least one target gene is a singletarget gene. In other embodiments, the at least one target gene ismultiple target genes. Suppression of a target gene includesnon-specific suppression, e.g., constitutive expression, as well asspecific expression, e.g., spatially specific, temporally specific,developmentally specific, or inducible gene suppression. Specificity ofsuppression of the at least one target gene is achieved by techniquesknown to those skilled in the art, such as by selecting a promoterhaving the desired specific expression pattern, or by selecting amicroRNA recognition site that is recognized by a mature miRNA havingthe desired specific expression pattern.

Transcription of the recombinant DNA construct is carried out by meansknown in the art. In some embodiments, transcription is constitutive ornon-specific, e.g., under the control of a constitutive promoter. Inother embodiments, transcription occurs under specific spatial,temporal, or inducible conditions. For example, the recombinant DNAconstruct can include a spatially, temporally, or inducible specificpromoter. In another example, the recombinant DNA construct can includea riboswitch (DNA that transcribes to an RNA aptamer capable of bindingto a ligand, and DNA that transcribes to regulatory RNA capable ofregulating expression of the target gene, wherein the regulation isdependent on the conformation of the regulatory RNA, and theconformation of the regulatory RNA is allosterically affected by thebinding state of the RNA aptamer) thereby allowing transcription of therecombinant DNA construct to be controlled by the binding state of theRNA aptamer and thus the presence (or absence) of the ligand.

This invention further provides a method of concurrently effecting genesuppression of at least one target gene and gene expression of at leastone gene of interest, including the steps of: (a) providing anon-natural transgenic plant including a regenerated plant prepared fromthe non-natural transgenic plant cell of this invention, or a progenyplant of the regenerated plant (as described above under the heading“Transgenic Plant Cells and Plants”), wherein the recombinant DNAconstruct further includes a gene expression element for expressing theat least one gene of interest; and (b) transcribing the recombinant DNAconstruct in the non-natural transgenic plant, wherein, when therecombinant DNA construct is transcribed in the non-natural transgenicplant, transcribed RNA that is capable of suppressing the at least onetarget gene and transcribed RNA encoding the at least one gene ofinterest are produced, whereby the at least one target gene issuppressed relative to its expression in the absence of transcription ofthe recombinant DNA construct and the at least one gene of interest isconcurrently expressed.

A gene of interest can include any coding or non-coding sequence fromany species (including, but not limited to, non-eukaryotes such asbacteria, and viruses; fungi; plants, including monocots and dicots,such as crop plants, ornamental plants, and non-domesticated or wildplants; invertebrates such as arthropods, annelids, nematodes, andmolluscs; and vertebrates such as amphibians, fish, birds, and mammals.Non-limiting examples of a non-coding sequence to be expressed by a geneexpression element include, but not limited to, 5′ untranslated regions,promoters, enhancers, or other non-coding transcriptional regions, 3′untranslated regions, terminators, intron, microRNAs, microRNA precursorDNA sequences, small interfering RNAs, RNA components of ribosomes orribozymes, small nucleolar RNAs, RNA aptamers capable of binding to aligand, and other non-coding RNAs. Non-limiting examples of a gene ofinterest further include, but are not limited to, translatable (coding)sequence, such as genes encoding transcription factors and genesencoding enzymes involved in the biosynthesis or catabolism of moleculesof interest (such as amino acids, fatty acids and other lipids, sugarsand other carbohydrates, biological polymers, and secondary metabolitesincluding alkaloids, terpenoids, polyketides, non-ribosomal peptides,and secondary metabolites of mixed biosynthetic origin). A gene ofinterest can be a gene native to the cell (e.g., a plant cell) in whichthe recombinant DNA construct of the invention is to be transcribed, orcan be a non-native gene. A gene of interest can be a marker gene, forexample, a selectable marker gene encoding antibiotic, antifungal, orherbicide resistance, or a marker gene encoding an easily detectabletrait (e.g., in a plant cell, phytoene synthase or other genes impartinga particular pigment to the plant), or a gene encoding a detectablemolecule, such as a fluorescent protein, luciferase, or a uniquepolypeptide or nucleic acid “tag” detectable by protein or nucleic aciddetection methods, respectively). Selectable markers are genes ofinterest of particular utility in identifying successful processing ofconstructs of the invention. Genes of interest include those genes alsodescribed above as target genes, under the heading “Target Genes”.

The gene of interest to be expressed by the gene expression element caninclude at least one gene selected from the group consisting of aeukaryotic target gene, a non-eukaryotic target gene, and a microRNAprecursor DNA sequence. The gene of interest can include a single geneor multiple genes (such as multiple copies of a single gene, multiplealleles of a single gene, or multiple genes including genes frommultiple species). In one embodiment, the gene expression element caninclude self-hydrolyzing peptide sequences, e.g., located betweenmultiple sequences coding for one or more polypeptides (see, forexample, the 2A and “2A-like” self-cleaving sequences from variousspecies, including viruses, trypanosomes, and bacteria, disclosed byDonnelly et al. (2001), J. Gen. Virol., 82:1027-1041). In anotherembodiment, the gene expression element can include ribosomal “skip”sequences, e.g., located between multiple sequences coding for one ormore polypeptides (see, for example, the aphthovirus foot-and-mouthdisease virus (FMDV) 2A ribosomal “skip” sequences disclosed by Donnellyet al. (2001), J. Gen. Virol., 82:1013-1025).

Abiotic-Stress-Responsive MiRNAs

A further aspect of this invention is directed to miRNAs that exhibit anexpression pattern that is responsive to abiotic stress, for example, amiRNA that exhibits an expression pattern characterized by regulation ofthe miRNA by nutrient stress, a miRNA that exhibits an expressionpattern characterized by regulation of the miRNA by water stress, or amiRNA that exhibits an expression pattern characterized by regulation ofthe miRNA by temperature stress.

Examples 6-11 describe a novel miRNA that was identified in crop plantsand assigned the trivial name miRMON18, which exhibits an expressionpattern characterized by suppression of the miRNA under nutrient stress(i.e., nitrogen deficiency, phosphate deficiency, or both nitrogen andphosphate deficiency). The mature miRMON18 is a 21-nucleotide miRNA withthe sequence UUAGAUGACCAUCAGCAAACA and was cloned from rice (SEQ ID NO.393), maize (SEQ ID NO. 3227), and soybean (SEQ ID NO. 8742) small RNAlibraries. Precursor sequences were identified in rice (SEQ ID NO. 1763)and in maize (SEQ ID NO. 3936).

Recombinant DNA constructs of this invention are described in detailunder the heading “Recombinant DNA Constructs” above and are useful withany of the miRNAs disclosed herein, for example, a mature miRNA selectedfrom SEQ ID NOS. 1-1035, SEQ ID NOS. 2730-3921, SEQ ID NOS. 5498-6683,SEQ ID NOS. 8409-8560, SEQ ID NO 8742, SEQ ID NO. 8744, SEQ ID NOS.8812-8815, SEQ ID NO. 8845, and SEQ ID NO. 8850, or a mature miRNAderived from a plant miRNA precursor sequence selected from SEQ ID NOS.1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS.8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819.The description of recombinant DNA constructs of this invention alsoapplies generally to embodiments of this invention that are morespecifically directed to a miRNAs having a particular expressionpattern, such as a nutrient-stress-responsive plant miRNA (e.g.,miRMON18 and other miRNAs described in the Examples) as described inthis section. The following description is directed to miRMON18 but isalso applicable to other miRNAs regulated by abiotic stress, especiallya miRNAs that exhibits an expression pattern characterized bysuppression of the miRNA under nutrient stress, a miRNA that exhibits anexpression pattern characterized by suppression of the miRNA under waterstress, or a miRNA that exhibits an expression pattern characterized bysuppression of the miRNA under temperature stress; non-limiting examplesof miRNAs regulated by abiotic stress include miR399 and miR319.

Thus, this invention provides a recombinant DNA construct including atleast one transcribable DNA element for modulating the expression of atleast one target gene, wherein the at least one transcribable DNAelement is selected from the group consisting of: (a) a DNA element thattranscribes to an miRNA precursor with the fold-back structure of amiRMON18 precursor sequence selected from SEQ ID NO. 1763 and SEQ ID NO.3936 and is processed to a mature miRMON18 miRNA having the sequence ofUUAGAUGACCAUCAGCAAACA (SEQ ID NO. 393, SEQ ID NO. 3227, or SEQ ID NO.8742); (b) a DNA element that transcribes to an engineered miRNAprecursor derived from the fold-back structure of a miRMON18 precursorsequence selected from SEQ ID NO. 1763 and SEQ ID NO. 3936, wherein theengineered miRNA precursor includes a modified mature miRMON18 miRNA;(c) a DNA element that is located within or adjacent to a transgenetranscription unit and that is transcribed to RNA including a miRNArecognition site recognized by a mature miRNA derived from a miRMON18precursor sequence selected from SEQ ID NO. 1763 and SEQ ID NO. 3936;and (d) a DNA element for suppressing expression of an endogenous miRNAderived from a miRMON18 precursor sequence selected from SEQ ID NO. 1763and SEQ ID NO. 3936. These embodiments directed to miRMON18 aredescribed in more detail below.

(A) Expression of a Native miRMON18 Under Non-Native Conditions.

This invention provides a recombinant DNA construct including at leastone transcribable DNA element for modulating the expression of at leastone target gene, wherein the at least one transcribable DNA elementincludes a DNA element that transcribes to a miRNA precursor with thefold-back structure of a miRMON18 precursor sequence selected from SEQID NO. 1763 and SEQ ID NO. 3936 and is processed to a mature miRMON18miRNA having the sequence of SEQ ID NO. 393, SEQ ID NO. 3227, or SEQ IDNO. 8742, and the at least one target gene is an endogenous gene of aplant, and wherein expression of the recombinant DNA construct in theplant results in suppression of the at least one target gene. In onepreferred embodiment, the miRNA precursor includes a contiguous segmentof at least 90% of the nucleotides of the miRMON18 precursor sequence.Such constructs are especially useful for expression of miRMON18 in anexpression pattern other than the native miRMON18 expression pattern(e.g., in different tissues, at different times, or at different levelsof expression).

The miRMON18 precursor need not include all of the nucleotides containedin a miRMON18 precursor sequence selected from SEQ ID NO. 1763 and SEQID NO. 3936, but preferably includes a contiguous segment of at least80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%,or at least 98%, or at least 99% of the nucleotides of a miRMON18precursor sequence selected from SEQ ID NO. 1763 and SEQ ID NO. 3936. Ina preferred embodiment, the miRNA precursor includes a contiguoussegment of at least 90% of the nucleotides of a miRMON18 precursorsequence selected from SEQ ID NO. 1763 and SEQ ID NO. 3936. Regardlessof the specific nucleotide sequence employed, the miRMON18 precursorforms a fold-back structure that is identical or near-identical to thefold-back structure formed by amiRMON18 precursor sequence selected fromSEQ ID NO. 1763 and SEQ ID NO. 3936 and is processed in vivo by one ormore steps to a mature miRMON18 miRNA having the sequence of SEQ ID NO.393, SEQ ID NO. 3227, or SEQ ID NO. 8742.

In preferred embodiments, the at least one target gene is an endogenousgene of a plant that includes at least one miRMON18 recognition site(target site), and expression of the recombinant DNA construct in theplant results in suppression of the at least one target gene. Inpreferred embodiments, the at least one target gene is an endogenousgene of a plant, and thus expression of the recombinant DNA construct inthe plant results in suppression of the at least one target gene. Inpreferred embodiments, the recombinant DNA construct further includes apromoter other than a native miRMON18 promoter. This permits expressionof the mature miRMON18 miRNA under spatial or temporal or inducibleconditions under which it would not natively be expressed. For example,the recombinant DNA construct can be designed to include a constitutivepromoter and thus constitutively express a mature miRMON18 that has anexpression pattern characterized by suppression of the miRNA undernutrient stress (i.e., nitrogen deficiency, phosphate deficiency, orboth nitrogen and phosphate deficiency); this would result inconstitutive suppression of the miRMON18 target gene. In anotherexample, the recombinant DNA construct can be designed to include aninducible root-specific promoter and thus express a mature miRMON18 inroot upon induction; this would result in suppression of the miRMON18target gene in root tissue upon induction. Promoters that are usefulwith this recombinant DNA construct are described under the heading“Promoters”.

(B) Expression of an Engineered Mature miRNA Derived from miRMON18.

In another embodiment, the recombinant DNA construct includes at leastone transcribable DNA element for modulating the expression of at leastone target gene, wherein the at least one transcribable DNA element formodulating the expression of at least one target gene includes a DNAelement that transcribes to an engineered miRNA precursor derived fromthe fold-back structure of a miRMON18 precursor sequence selected fromSEQ ID NO. 1763 and SEQ ID NO. 3936, wherein the engineered miRNAprecursor includes a modified mature miRMON18 miRNA, wherein the atleast one target gene is an endogenous gene of a plant or an endogenousgene of a pest or pathogen of the plant, and wherein expression of therecombinant DNA construct in the plant results in suppression of the atleast one target gene.

In preferred embodiments, the at least one target gene is an endogenousgene of a plant or an endogenous gene of a pest or pathogen of theplant, and expression of the recombinant DNA construct in the plantresults in suppression of the at least one target gene. Suitable targetgenes are described above under the heading “Target Genes”. By“engineered” is meant that nucleotides are changed (substituted,deleted, or added) in a native miRMON18 precursor sequence selected fromSEQ ID NO. 1763 and SEQ ID NO. 3936, thereby resulting in an engineeredmiRNA precursor having substantially the same the fold-back structure asthe native miRMON18 precursor sequence, but wherein the mature miRNAthat is processed from the engineered miRMON18 precursor has a modifiedsequence (i.e., different from that of the native mature miRMON18) thatis designed to suppress a target gene different from the target genesnatively suppressed by the native miRMON18 precursor sequence. Ageneral, non-limiting method for determining nucleotide changes in thenative miRMON18 precursor sequence to produce the engineered miRNAprecursor is described above under the heading “Expression of anengineered mature miRNA”.

(C) Expression of a Transgene and a miRMON18 Recognition Site.

In another embodiment, the recombinant DNA construct includes at leastone transcribable DNA element for modulating the expression of at leastone target gene, and further includes a transgene transcription unit,wherein the at least one transcribable DNA element for modulating theexpression of at least one target gene includes a DNA element that islocated within or adjacent to the transgene transcription unit and thatis transcribed to RNA including a miRNA recognition site recognized by amature miRMON18 miRNA having the sequence of SEQ ID NO. 393, SEQ ID NO.3227, or SEQ ID NO. 8742 or by a mature miRMON18 miRNA derived from amiRMON18 precursor sequence selected from SEQ ID NO. 1763 and SEQ ID NO.3936, and the at least one target gene includes the transgene encoded bythe transgene transcription unit, and wherein expression of therecombinant DNA construct in a plant results in expression of thetransgene in cells of the plant wherein the mature miRMON18 miRNA is notnatively expressed. Prediction of a miRMON18 recognition site isachieved using methods known in the art, such as sequencecomplementarity rules as described by Zhang (2005) Nucleic Acids Res.,33:W701-704 and by Rhoades et al. (2002) Cell, 110:513-520; non-limitingexamples of miRMON18 recognition sites are provided in the workingExamples below.

Prediction of a miRMON18 recognition site permits identification andvalidation of endogenous genes regulated by a mature miRMON18 from anatively expressed miRMON18 precursor; this is useful, e.g., toeliminate or modify a miRMON18 recognition site in an endogenous gene inorder to decouple expression of that gene from regulation by theendogenous miRMON18 that natively regulates expression of the gene. Inone embodiment, the number of mismatches (especially those correspondingto positions 2 to 13 of the mature miRMON18) between a miRMON18recognition site and a mature miRMON18 can be increased to preventrecognition and cleavage by an endogenous miRMON18.

These recombinant DNA constructs are particularly useful for in plantaexpression of the transgene to be restricted according to the endogenousexpression of miRMON18, that is, the transgene is expressed whenmiRMON18 is suppressed, such as under nutrient stress (i.e., nitrogendeficiency, phosphate deficiency, or both nitrogen and phosphatedeficiency). Expression of the transgene can be further controlled byuse of an appropriate promoter. In a non-limiting example, a recombinantDNA construct of this invention that encodes (a) a transgene under thecontrol of a root-specific promoter and (b) a miRNA recognition siterecognized by a mature miRMON18 that is specifically suppressed onlyunder conditions of nitrogen (or phosphate) deficiency is used forexpression of the transgene in roots of a plant under nitrogen-deficient(or phosphate-deficient) conditions.

The transgene transcription unit includes at least a transgene, andoptionally additional sequence such as, but not limited to, a promoter,a promoter enhancer, a terminator, messenger RNA stabilizing ordestabilizing sequence (see, e.g., Newman et al. (1993) Plant Cell,5:701-714; Green (1993) Plant Physiol., 102:1065-1070; and Ohme-Takagiet al. (1993) Proc. Natl. Acad. Sci. USA, 90:11811-11815), sequence forlocalization or transport of the transgene transcript to a specificlocale (e.g., mitochondrion, plastid, nucleolus, peroxisome, endoplasmicreticulum, etc.), or other sequence related to the desired processing ofthe transgene. The transgene encoded by the transgene transcription unitcan include any one or more genes of interest, including codingsequence, non-coding sequence, or both. Genes of interest can includeany of the genes listed under “Target Genes”, preferred examples ofwhich include translatable (coding) sequence for genes encodingtranscription factors and genes encoding enzymes involved in thebiosynthesis or catabolism of molecules of interest (such as, but notlimited to, amino acids, fatty acids and other lipids, sugars and othercarbohydrates, biological polymers, and secondary metabolites includingalkaloids, terpenoids, polyketides, non-ribosomal peptides, andsecondary metabolites of mixed biosynthetic origin).

(D) Suppression of an Endogenous or Native miRMON18.

In another embodiment, the recombinant DNA construct includes at leastone transcribable DNA element for modulating the expression of at leastone target gene, wherein the at least one transcribable DNA elementincludes a DNA element for suppressing expression of an endogenousmature miRMON18 miRNA derived from a miRMON18 precursor sequenceselected from SEQ ID NO. 1763 and SEQ ID NO. 3936, wherein the at leastone target gene is an endogenous gene of a plant, and wherein expressionof the endogenous gene is suppressed in cells of the plant where nativeexpression of the endogenous mature miRMON18 miRNA occurs, and whereinexpression of the recombinant DNA construct in the cells results inexpression of the endogenous gene in the cells. Such constructs areespecially useful for suppression of a native or endogenous miRMON18 andthus for permitting expression of genes that have one or more miRMON18recognition sites. In preferred embodiments, the at least one targetgene is an endogenous gene of a plant and includes one or more miRMON18recognition sites, and expression of the endogenous gene is suppressedin cells of the plant where native expression of the mature miRMON18occurs, and thus expression of the recombinant DNA construct in thecells results in expression of the endogenous target gene in the cells.

The DNA element for suppressing expression includes at least one of:

-   -   (a) DNA that includes at least one anti-sense DNA segment that        is anti-sense to at least one segment of the target gene;    -   (b) DNA that includes multiple copies of at least one anti-sense        DNA segment that is anti-sense to at least one segment of the        target gene;    -   (c) DNA that includes at least one sense DNA segment that is at        least one segment of the target gene;    -   (d) DNA that includes multiple copies of at least one sense DNA        segment that is at least one segment of the target gene;    -   (e) DNA that transcribes to RNA for suppressing the target gene        by forming double-stranded RNA and includes at least one        anti-sense DNA segment that is anti-sense to at least one        segment of the target gene and at least one sense DNA segment        that is at least one segment of the target gene;    -   (f) DNA that transcribes to RNA for suppressing the target gene        by forming a single double-stranded RNA and includes multiple        serial anti-sense DNA segments that are anti-sense to at least        one segment of the target gene and multiple serial sense DNA        segments that are at least one segment of the target gene;    -   (g) DNA that transcribes to RNA for suppressing the target gene        by forming multiple double strands of RNA and includes multiple        anti-sense DNA segments that are anti-sense to at least one        segment of the target gene and multiple sense DNA segments that        are at least one segment of the target gene, and wherein the        multiple anti-sense DNA segments and the multiple sense DNA        segments are arranged in a series of inverted repeats;    -   (h) DNA that includes nucleotides derived from a plant miRNA;    -   (i) DNA that includes nucleotides of a siRNA;    -   (j) DNA that transcribes to an RNA aptamer capable of binding to        a ligand; and    -   (k) DNA that transcribes to an RNA aptamer capable of binding to        a ligand, and DNA that transcribes to regulatory RNA capable of        regulating expression of the target gene, wherein the regulation        is dependent on the conformation of the regulatory RNA, and the        conformation of the regulatory RNA is allosterically affected by        the binding state of the RNA aptamer.

DNA elements for suppressing expression are described further in Example3 and depicted in FIGS. 2 and 3. The effects of a miRNA on its targetgene can also be controlled by alternative methods described in detailbelow under “MicroRNA Decoy Sequences”.

In some embodiments, the recombinant DNA construct includes DNA designedto be transcribed to single-stranded RNA or to at least partiallydouble-stranded RNA (such as in a “kissing stem-loop” arrangement), orto an RNA that assumes a secondary structure or three-dimensionalconfiguration (e.g., a large loop of antisense sequence of the targetgene or an aptamer) that confers on the transcript an additional desiredcharacteristic, such as increased stability, increased half-life invivo, or cell or tissue specificity. In one example, the spacer istranscribed to a stabilizing loop that links the first and second seriesof contiguous RNA segments (see, for example, Di Giusto and King (2004)J. Biol. Chem., 279:46483-46489). In another example, the recombinantDNA construct includes DNA that transcribes to RNA including an RNAaptamer (e.g., an aptamer that binds to a cell-specific ligand) thatallows cell- or tissue-specific targetting of the recombinant RNAduplex.

(E) miRNA-Unresponsive Transgenes, Including miRMON18-UnresponsiveTransgenes.

Also disclosed and claimed is a recombinant DNA construct including asynthetic miRNA-unresponsive transgene sequence that is unresponsive toa given mature miRNA, wherein the synthetic miRNA-unresponsive transgenesequence is: (a) derived from a natively miRNA-responsive sequence bydeletion or modification of all native miRNA recognition sitesrecognized by the given mature miRNA within the nativelymiRNA-responsive sequence, and (b) is not recognized by the given maturemiRNA. Non-limiting embodiments include a recombinant DNA constructincluding a synthetic miRNA-unresponsive transgene sequence that isunresponsive to a mature miRNA selected from SEQ ID NOS. 1-1035, SEQ IDNOS. 2730-3921, SEQ ID NOS. 5498-6683, SEQ ID NOS. 8409-8560, SEQ ID NO8742, SEQ ID NO. 8744, SEQ ID NOS. 8812-8815, SEQ ID NO. 8845, and SEQID NO. 8850, or unresponsive to a mature miRNA derived from a plantmiRNA precursor sequence selected from SEQ ID NOS. 1036-2690, SEQ IDNOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO.8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819, wherein the syntheticmiRNA-unresponsive transgene sequence is: (a) derived from a nativelymiRNA-responsive sequence by deletion or modification of all nativemiRNA recognition sites recognized by the given mature miRNA within thenatively miRNA-responsive sequence, and (b) is not recognized by thegiven mature miRNA. Prediction of a recognition site is achieved usingmethods known in the art, such as sequence complementarity rules asdescribed by Zhang (2005) Nucleic Acids Res., 33:W701-704 and by Rhoadeset al. (2002) Cell, 110:513-520.

One non-limiting preferred embodiment is a recombinant DNA constructincluding a synthetic miRMON18-unresponsive transgene sequence, whereinthe synthetic miRMON18-unresponsive transgene sequence is: (a) derivedfrom a natively miRMON18-responsive sequence by deletion or modificationof all native miRMON18 miRNA recognition sites (that is to say, deletionor modification of any recognition site that is recognized by a maturemiRMON18 miRNA having the sequence of SEQ ID NO. 393, SEQ ID NO. 3227,or SEQ ID NO. 8742 or by a mature miRMON18 miRNA derived from a miRMON18precursor sequence selected from SEQ ID NO. 1763 and SEQ ID NO. 3936)within the natively miRMON18-responsive sequence, and (b) is notrecognized by a mature miRMON18 miRNA.

(F) Abiotic-Stress-Responsive miRNA Promoters, Including miRMON18Promoters.

Also disclosed and claimed is a recombinant DNA construct including apromoter of a miRNA that exhibits an expression pattern characterized byregulation by abiotic stress, for example, a promoter of a miRNA thatexhibits an expression pattern characterized by regulation of the miRNAby nutrient stress, a promoter of a miRNA that exhibits an expressionpattern characterized by regulation of the miRNA by water stress, or apromoter of a miRNA that exhibits an expression pattern characterized byregulation of the miRNA by temperature stress. Preferred embodimentsinclude a recombinant DNA construct including a promoter of a miRNA thatexhibits an expression pattern characterized by regulation of the miRNAby nutrient stress, wherein the nutrient stress comprises at least onenutrient deficiency selected from the group consisting of nitrogendeficiency and phosphate deficiency. In one embodiment, the promoter isthat of a miRNA that is suppressed by nitrogen deficiency. In anotherembodiment, the promoter is that of a miRNA that is suppressed byinorganic phosphate deficiency. In yet another embodiment, the promoteris that of a miRNA that is suppressed by the co-occurrence of nitrogenand phosphate deficiency. In further embodiments, the promoter is thatof a miRNA that is upregulated by by nitrogen deficiency or by phosphatedeficiency.

Particularly preferred embodiments include a recombinant DNA constructincluding a promoter of a miRNA that exhibits an expression patterncharacterized by suppression of the miRNA under nutrient stress, whereinthe nutrient stress comprises at least one nutrient deficiency selectedfrom the group consisting of nitrogen deficiency and phosphatedeficiency, and wherein the promoter includes at least one of: (a) thepromoter of a maize miRNA that exhibits in leaf tissue strong expressionunder nitrogen-sufficient conditions and suppression undernitrogen-deficient conditions; (b) the promoter of a maize miRNA thatexhibits in leaf tissue strong expression under phosphate-sufficientconditions and suppression under phosphate-deficient conditions; (c) amiRMON18 promoter having the sequence of SEQ ID NO. 8804; (d) a fragmentof at least about 50 contiguous nucleotides having at least 85% identityto a segment of SEQ ID NO. 8804. Also preferred are embodiments whereinthe promoter is operably linked to at least one of: (a) a genesuppression element, and (b) a gene expression element; preferably,these embodiments are useful for expressing the recombinant DNAconstruct in a plant

Non-limiting examples include the promoter having the sequence ofnucleotides 211-2172 of SEQ ID NO. 8800; a fragment of at least about50, at least about 100, at least about 150, at least about 200, at leastabout 300, at least about 400, or at least 500 contiguous nucleotideshaving at least 85%, at least 90%, at least 95%, or at least 98%identity to nucleotides 211-2172 of SEQ ID NO. 8800, wherein thefragment has promoter activity in at least one plant tissue that ischaracterized by strong expression under nitrogen-sufficient conditionsand suppression under nitrogen-deficient conditions or strong expressionunder phosphate-sufficient conditions and suppression underphosphate-deficient conditions; and a fragment of at least about 50, atleast about 100, at least about 150, at least about 200, at least about300, at least about 400, or at least 500 contiguous nucleotides havingat least 85%, at least 90%, at least 95%, or at least 98% identity toSEQ ID NO. 8804, wherein the fragment has promoter activity in at leastone plant tissue that is characterized by strong expression undernitrogen-sufficient conditions and suppression under nitrogen-deficientconditions or strong expression under phosphate-sufficient conditionsand suppression under phosphate-deficient conditions.

(G) Abiotic-Stress-Responsive Transgenic Plant Cells and Plants

Further disclosed and claimed is a non-natural transgenic plant cellincluding any of the recombinant DNA constructs disclosed under thisheading (“Abiotic-Stress-Responsive miRNAs”). One preferred embodimentincludes a non-natural transgenic plant prepared from a non-naturaltransgenic plant cell including a recombinant DNA construct including atleast one transcribable DNA element for modulating the expression of atleast one target gene, wherein the at least one transcribable DNAelement includes a DNA element that transcribes to an miRNA precursorwith the fold-back structure of a miRMON18 precursor sequence selectedfrom SEQ ID NO. 1763, SEQ ID NO. 3936, and SEQ ID NO. 8800, wherein themiRNA precursor includes a contiguous segment of at least 90% of thenucleotides of the miRMON18 precursor sequence and is processed to amature miRMON18 miRNA having the sequence of UUAGAUGACCAUCAGCAAACA (SEQID NO. 393, SEQ ID NO. 3227, or SEQ ID NO. 8742) and the at least onetarget gene is an endogenous gene of a plant and includes an SPX domain,and wherein expression of the recombinant DNA construct in the plantresults in suppression of the at least one target gene; generally therecombinant DNA construct further includes a promoter other than thenative miRMON18 promoter to drive expression of the mature miRMON18.

Another preferred embodiment includes a non-natural transgenic plantprepared from a non-natural transgenic plant cell including arecombinant DNA construct including at least one transcribable DNAelement for modulating the expression of at least one target gene,wherein the at least one transcribable DNA element includes a DNAelement for suppressing expression of an endogenous mature miRMON18miRNA derived from a miRMON18 precursor sequence selected from SEQ IDNO. 1763, SEQ ID NO. 3936, and SEQ ID NO. 8800, the at least one targetgene is an endogenous gene of a plant and includes an SPX domain, andexpression of the endogenous gene is suppressed in cells of the plantwhere native expression of the endogenous mature miRMON18 miRNA occurs,and wherein expression of the recombinant DNA construct in the cellsresults in expression of the endogenous gene in the cells. Suitable DNAelements for suppressing expression of an endogenous mature miRMON18miRNA are described above under the heading “Suppression of anendogenous or native miRMON18”.

MicroRNA Decoy Sequences

Plant microRNAs regulate their target genes by recognizing and bindingto a near-perfectly complementary sequence (miRNA recognition site) inthe target transcript, followed by cleavage of the transcript by RNaseIII enzymes such as Ago1. In plants, certain mismatches between a givenmiRNA recognition site and the corresponding mature miRNA are nottolerated, particularly mismatched nucleotides at positions 10 and 11 ofthe mature miRNA. Positions within the mature miRNA are given in the 5′to 3′ direction; for clarity, FIG. 7D depicts examples of miRNAs, miR827(SEQ ID NO. 8744) and miRMON18 (SEQ ID NO. 393, SEQ ID NO. 3227, or SEQID NO. 8742), with numbered arrows indicating positions 1, 10, and 21 ofthe mature miRNA; the nucleotide at position 10 is also underlined.Perfect complementarity between a given miRNA recognition site and thecorresponding mature miRNA is usually required at positions 10 and 11 ofthe mature miRNA. See, for example, Franco-Zorrilla et al. (2007) NatureGenetics, 39:1033-1037; and Axtell et al. (2006) Cell, 127:565-577.

This characteristic of plant miRNAs was exploited to arrive at rules forpredicting a “microRNA decoy sequence”, i.e., a sequence that can berecognized and bound by an endogenous mature miRNA resulting inbase-pairing between the miRNA decoy sequence and the endogenous maturemiRNA, thereby forming a cleavage-resistant RNA duplex that is notcleaved because of the presence of mismatches between the miRNA decoysequence and the mature miRNA. Mismatches include canonical mismatches(e.g., G-A, C-U, C-A) as well as G::U wobble pairs and indels(nucleotide insertions or deletions). In general, these rules define (1)mismatches that are required, and (2) mismatches that are permitted butnot required.

Required mismatches include: (a) at least 1 mismatch between the miRNAdecoy sequence and the endogenous mature miRNA at positions 9, 10, or 11of the endogenous mature miRNA, or alternatively, (b) 1, 2, 3, 4, or 5insertions (i.e., extra nucleotides) at a position in the miRNA decoysequence corresponding to positions 9, 10, or 11 of the endogenousmature miRNA. In preferred embodiments, there exists either (a) at least1 mismatch between the miRNA decoy sequence and the endogenous maturemiRNA at positions 10 and/or 11 of the endogenous mature miRNA, or (b)at least 1 insertion at a position in the miRNA decoy sequencecorresponding to positions 10 and/or 11 of the endogenous mature miRNA.

Mismatches that are permitted, but not required, include: (a) 0, 1, or 2mismatches between the miRNA decoy sequence and the endogenous maturemiRNA at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of the endogenousmature miRNA, and (b) 0, 1, 2, or 3 mismatches between the miRNA decoysequence and the endogenous mature miRNA at positions 12 through thelast position of the endogenous mature miRNA (i.e., at position 21 of a21-nucleotide mature miRNA), wherein each of the mismatches at positions12 through the last position of the endogenous mature miRNA is adjacentto at least one complementary base-pair (i.e., so that there is not morethan 2 contiguous mismatches at positions 12 through the last positionof the endogenous mature miRNA). In preferred embodiments, there existno mismatches (i.e., there are all complementary base-pairs) atpositions 1, 2, 3, 4, 5, 6, 7, and 8 of the endogenous mature miRNA.

The miRNA decoy sequence can be of any length as long as it isrecognized and bound by an endogenous mature miRNA to form acleavage-resistant RNA duplex. In preferred embodiments, the miRNA decoysequence includes between about 18 to about 36 nucleotides. Specificallyclaimed embodiments include miRNA decoy sequences of 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, and 31 nucleotides. In non-limitingexamples, a miRNA decoy sequence (for a 21-nucleotide mature miRNA)having a required mismatch consisting of a 4-nucleotide insertion atposition 10 of the mature miRNA and a permitted mismatch consisting of a1-nucleotide insertion at position 20 of the mature miRNA has a total of26 nucleotides; a miRNA decoy sequence (for a 25-nucleotide maturemiRNA) having a required mismatch consisting of a 5-nucleotide insertionat position 11 of the mature miRNA and permitted mismatches consistingof a canonical mismatch at position 20 of the mature miRNA and1-nucleotide insertion at position 23 of the mature miRNA will have atotal of 31 nucleotides.

Thus, one embodiment of this invention includes a recombinant DNAconstruct that is transcribed to an RNA transcript including at leastone miRNA decoy sequence that is recognized and bound by an endogenousmature miRNA but not cleaved (e.g., not cleaved by Argonaute or anAGO-like protein), wherein the endogenous miRNA is at least one miRNAselected from (a) mature miRNA selected from a mature miRNA selectedfrom SEQ ID NOS. 1-1035, SEQ ID NOS. 2730-3921, SEQ ID NOS. 5498-6683,SEQ ID NOS. 8409-8560, SEQ ID NO 8742, SEQ ID NO. 8744, SEQ ID NOS.8812-8815, SEQ ID NO. 8845, and SEQ ID NO. 8850, or (b) a mature miRNAderived from a plant miRNA precursor sequence selected from SEQ ID NOS.1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS.8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819;and the miRNA decoy sequence includes an RNA sequence of between about19 to about 36 contiguous RNA nucleotides, wherein the miRNA decoysequence is recognized and bound by the endogenous mature miRNA,resulting in base-pairing between the miRNA decoy sequence and theendogenous mature miRNA, thereby forming a cleavage-resistant RNA duplexincluding: (a) at least one mismatch between said miRNA decoy sequenceand said endogenous mature miRNA at positions 9, 10, or 11 of saidendogenous mature miRNA, or at least one insertion at a position in saidmiRNA decoy sequence corresponding to positions 10-11 of said endogenousmature miRNA, (b) 0, 1, or 2 mismatches between said miRNA decoysequence and said endogenous mature miRNA at positions 1, 2, 3, 4, 5, 6,7, 8, and 9 of said endogenous mature miRNA, and (c) 0, 1, 2, or 3mismatches between said miRNA decoy sequence and said endogenous maturemiRNA at positions 12 through the last position of said endogenousmature miRNA, wherein each of said mismatches at positions 12 throughthe last position of said endogenous mature miRNA is adjacent to atleast one complementary base-pair.

Recombinant DNA constructs of this invention include at least one miRNAdecoy sequence, and can include multiple miRNA decoy sequences (eithermultiple copies of a single miRNA decoy sequence, or copies of differentmiRNA decoy sequences, or a combination of both). In one example,multiple copies of a miRNA decoy sequence are arranged in tandem in arecombinant DNA construct designed to decrease the activity of thecorresponding mature miRNA. In another example, the activity ofdifferent mature miRNAs is decreased by expressing a single chimericrecombinant DNA construct that transcribes to multiple different miRNAdecoy sequences. Expression of miRNA decoy sequences can be driven byvarious promoters, including, but not limited to, tissue-specific,cell-specific, temporally specific, inducible, or constitutivepromoters, for example, any of the promoters described under the heading“Promoters”. The miRNA decoy sequences can be located in variouspositions in a transcript. In a recombinant DNA construct that isintended to also transcribe to coding sequence, non-coding sequence(e.g., a miRNA), or both, the miRNA decoy sequence is preferably locatedin an intron or after the polyadenylation signal, to permit normaltranscription of the coding sequence, non-coding sequence, or both.

In further embodiments of this invention, analogous decoy sequences areused to regulate the activity of other small RNAs involved indouble-stranded RNA-mediated gene suppression, including trans-actingsmall interfering RNAs (ta-siRNAs), natural anti-sense transcript siRNAs(nat-siRNAs), and phased small RNAs (as described in U.S. patentapplication Ser. No. 11/897,611, filed 31 Aug. 2007, which isincorporated by reference herein). These analogous ta-siRNA decoysequences, nat-siRNAs decoy sequences, and phased small RNA decoysequences are predicted using essentially the same rules as those forpredicting miRNA decoy sequences, and have utilities similar to those ofthe miRNA decoy sequences.

The miRNA decoy sequence can be a naturally-occurring sequence or anartificial sequence. In one embodiment, the at least one miRNA decoysequence includes a naturally occurring miRNA decoy sequence, forexample, an endogenous miRNA decoy sequence identified bybioinformatics. In another embodiment the at least one miRNA decoysequence includes a synthetic miRNA decoy sequence, for example, onethat is designed ab initio to bind to a given mature miRNA to form acleavage-resistant RNA duplex.

Thus, one embodiment of this invention is a recombinant DNA constructthat is transcribed to an RNA transcript including at least one miRMON18decoy sequence that is recognized and bound by an endogenous maturemiRMON18 but not cleaved (e.g., not cleaved by Argonaute or an AGO-likeprotein), wherein the endogenous miRMON18 is at least one selected from(a) a mature miRMON18, or (b) a mature miRNA derived from a plantmiRMON18 precursor sequence; and the miRMON18 decoy sequence includes anRNA sequence of between about 19 to about 36 contiguous RNA nucleotides,wherein the miRMON18 decoy sequence is recognized and bound by theendogenous mature miRMON18, resulting in base-pairing between themiRMON18 decoy sequence and the endogenous mature miRMON18, therebyforming a cleavage-resistant RNA duplex including: (a) at least onemismatch between the miRMON18 decoy sequence and the endogenous maturemiRMON18 at positions 9, 10, or 11 of the endogenous mature miRMON18, orat least one insertion at a position in the miRMON18 decoy sequencecorresponding to positions 10-11 of the endogenous mature miRMON18, (b)0, 1, or 2 mismatches between the miRMON18 decoy sequence and theendogenous mature miRMON18 at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 ofthe endogenous mature miRMON18, and (c) 0, 1, 2, or 3 mismatches betweenthe miRMON18 decoy sequence and the endogenous mature miRMON18 atpositions 12 through the last position of the endogenous maturemiRMON18, wherein each of the mismatches at positions 12 through thelast position of the endogenous mature miRMON18 is adjacent to at leastone complementary base-pair; and wherein the at least one miRMON18 decoysequence is recognized and bound but not cleaved by a mature miRMON18miRNA. In preferred embodiments, the mature miRMON18 has the sequence ofSEQ ID NO. 393, SEQ ID NO. 3227, or SEQ ID NO. 874, or is a mature miRNAderived from a miRMON18 precursor sequence selected from SEQ ID NO. 1763and SEQ ID NO. 3936. Further provided by this invention is a method ofproviding a non-natural transgenic crop plant having improved yieldunder at least one nutrient deficiency selected from nitrogen deficiencyand phosphate deficiency, including expressing in the non-naturaltransgenic crop plant a recombinant DNA construct that is transcribed toan RNA transcript including at least one miRMON18 decoy sequence.

Another embodiment of this invention is a recombinant DNA construct thatis transcribed to an RNA transcript including at least one miR399 decoysequence that is recognized and bound by an endogenous mature miR399 butnot cleaved (e.g., not cleaved by Argonaute or an AGO-like protein),wherein the endogenous miR399 is at least one selected from (a) a maturemiR399, or (b) a mature miRNA derived from a miR399 precursor sequenceselected from SEQ ID NOS. 8816-8819; and the miR399 decoy sequenceincludes an RNA sequence of between about 19 to about 36 contiguous RNAnucleotides, wherein the miR399 decoy sequence is recognized and boundby the endogenous mature miR399, resulting in base-pairing between themiR399 decoy sequence and the endogenous mature miR399, thereby forminga cleavage-resistant RNA duplex including: (a) at least one mismatchbetween the miR399 decoy sequence and the endogenous mature miR399 atpositions 9, 10, or 11 of the endogenous mature miR399, or at least oneinsertion at a position in the miR399 decoy sequence corresponding topositions 10-11 of the endogenous mature miR399, (b) 0, 1, or 2mismatches between the miR399 decoy sequence and the endogenous maturemiR399 at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of the endogenousmature miR399, and (c) 0, 1, 2, or 3 mismatches between the miR399 decoysequence and the endogenous mature miR399 at positions 12 through thelast position of the endogenous mature miR399, wherein each of themismatches at positions 12 through the last position of the endogenousmature miR399 is adjacent to at least one complementary base-pair; andwherein the at least one miR399 decoy sequence is recognized and boundbut not cleaved by a mature miR399. In preferred embodiments, the maturemiR399 has the sequence of SEQ ID NOS. 8812-8815 or is a mature miRNAderived from a miR399 precursor sequence selected from SEQ ID NOS.8816-8819. Further provided by this invention is a method of providing anon-natural transgenic crop plant having improved yield under at leastone nutrient deficiency selected from nitrogen deficiency and phosphatedeficiency, including expressing in the non-natural transgenic cropplant a recombinant DNA construct that is transcribed to an RNAtranscript including at least one miR399 decoy sequence.

Yet another embodiment of this invention is suppression of an endogenousmiRNA decoy sequence, for example, by means of a gene suppressionelement (such as those described under the header “DNA element forsuppressing expression”), especially driven by a cell- ortissue-specific or an inducible promoter.

Any of these recombinant DNA constructs described herein can be made bycommonly used techniques, such as those described under the heading“Making and Using Recombinant DNA Constructs” and illustrated in theworking Examples. The recombinant DNA constructs are particularly usefulfor making non-natural transgenic plant cells, non-natural transgenicplants, and transgenic seeds as discussed below under “Transgenic PlantCells and Transgenic Plants”.

Recombinant DNA constructs including a miRNA decoy sequence are usefulfor providing unique expression patterns for a synthetic miRNA that isengineered to suppress an endogenous gene; this is especially desirablefor preventing adverse phenotypes caused by undesirable expression ofthe synthetic miRNA in certain tissues. For example, the synthetic miRNAcan be used to suppress the endogenous gene only in specific tissues ofa plant, e.g., by expression in the plant of a recombinant DNA constructincluding (a) a constitutive promoter driving expression of thesynthetic miRNA, and (b) a tissue-specific promoter driving expressionof a miRNA decoy sequence designed to sequester the synthetic miRNA.

Further provided by this invention are methods useful in providingimproved crop plants. One aspect of this invention includes a method ofproviding a non-natural transgenic crop plant having at least onealtered trait including expressing in the non-natural transgenic cropplant a recombinant DNA construct that is transcribed to an RNAtranscript including at least one miRNA decoy sequence that isrecognized and bound by an endogenous mature miRNA but not cleaved(e.g., not cleaved by Argonaute or an AGO-like protein), wherein theendogenous miRNA is at least one miRNA selected from (a) a mature miRNAselected from a mature miRNA selected from SEQ ID NOS. 1-1035, SEQ IDNOS. 2730-3921, SEQ ID NOS. 5498-6683, SEQ ID NOS. 8409-8560, SEQ ID NO8742, SEQ ID NO. 8744, SEQ ID NOS. 8812-8815, SEQ ID NO. 8845, and SEQID NO. 8850, or (b) a mature miRNA derived from a plant miRNA precursorsequence selected from SEQ ID NOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO.8800, and SEQ ID NOS. 8816-8819; and the miRNA decoy sequence includesan RNA sequence of between about 19 to about 36 contiguous RNAnucleotides, wherein the miRNA decoy sequence is recognized and bound bythe endogenous mature miRNA, resulting in base-pairing between the miRNAdecoy sequence and the endogenous mature miRNA, thereby forming acleavage-resistant RNA duplex including: (a) at least one mismatchbetween said miRNA decoy sequence and said endogenous mature miRNA atpositions 9, 10, or 11 of said endogenous mature miRNA, or at least oneinsertion at a position in said miRNA decoy sequence corresponding topositions 10-11 of said endogenous mature miRNA, (b) 0, 1, or 2mismatches between said miRNA decoy sequence and said endogenous maturemiRNA at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of said endogenousmature miRNA, and (c) 0, 1, 2, or 3 mismatches between said miRNA decoysequence and said endogenous mature miRNA at positions 12 through thelast position of said endogenous mature miRNA, wherein each of saidmismatches at positions 12 through the last position of said endogenousmature miRNA is adjacent to at least one complementary base-pair,thereby resulting in the non-natural transgenic crop plant exhibiting atleast one altered trait, relative to a crop plant not expressing therecombinant DNA construct, selected from the group of traits consistingof:

-   -   (i) improved abiotic stress tolerance;    -   (ii) improved biotic stress tolerance;    -   (iii) improved resistance to a pest or pathogen of the plant;    -   (iv) modified primary metabolite composition;    -   (v) modified secondary metabolite composition;    -   (vi) modified trace element, carotenoid, or vitamin composition;    -   (vii) improved yield;    -   (viii) improved ability to use nitrogen or other nutrients;    -   (ix) modified agronomic characteristics;    -   (x) modified growth or reproductive characteristics; and    -   (xi) improved harvest, storage, or processing quality.

In another aspect, this invention provides a method of providing anon-natural transgenic crop plant having at least one altered traitincluding suppressing in the non-natural transgenic crop plant at leastone endogenous miRNA decoy sequence that is recognized and bound by anendogenous mature miRNA but not cleaved (e.g., not cleaved by Argonauteor an AGO-like protein), wherein the endogenous miRNA is at least onemiRNA selected from (a) a mature miRNA selected from a mature miRNAselected from SEQ ID NOS. 1-1035, SEQ ID NOS. 2730-3921, SEQ ID NOS.5498-6683, SEQ ID NOS. 8409-8560, SEQ ID NO 8742, SEQ ID NO. 8744, SEQID NOS. 8812-8815, SEQ ID NO. 8845, and SEQ ID NO. 8850, or (b) a maturemiRNA derived from a plant miRNA precursor sequence selected from SEQ IDNOS. 1036-2690, SEQ ID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ IDNOS. 8561-8417, SEQ ID NO. 8743, SEQ ID NO. 8800, and SEQ ID NOS.8816-8819; and the miRNA decoy sequence includes an RNA sequence ofbetween about 19 to about 36 contiguous RNA nucleotides, wherein themiRNA decoy sequence is recognized and bound by the endogenous maturemiRNA, resulting in base-pairing between the miRNA decoy sequence andthe endogenous mature miRNA, thereby forming a cleavage-resistant RNAduplex including: (a) at least one mismatch between said miRNA decoysequence and said endogenous mature miRNA at positions 9, 10, or 11 ofsaid endogenous mature miRNA, or at least one insertion at a position insaid miRNA decoy sequence corresponding to positions 10-11 of saidendogenous mature miRNA, (b) 0, 1, or 2 mismatches between said miRNAdecoy sequence and said endogenous mature miRNA at positions 1, 2, 3, 4,5, 6, 7, 8, and 9 of said endogenous mature miRNA, and (c) 0, 1, 2, or 3mismatches between said miRNA decoy sequence and said endogenous maturemiRNA at positions 12 through the last position of said endogenousmature miRNA, wherein each of said mismatches at positions 12 throughthe last position of said endogenous mature miRNA is adjacent to atleast one complementary base-pair; thereby resulting in the non-naturaltransgenic crop plant exhibiting at least one altered trait, relative toa crop plant not expressing the recombinant DNA construct, selected fromthe group of traits consisting of:

-   -   (i) improved abiotic stress tolerance;    -   (ii) improved biotic stress tolerance;    -   (iii) improved resistance to a pest or pathogen of the plant;    -   (iv) modified primary metabolite composition;    -   (v) modified secondary metabolite composition;    -   (vi) modified trace element, carotenoid, or vitamin composition;    -   (vii) improved yield;    -   (viii) improved ability to use nitrogen or other nutrients;    -   (ix) modified agronomic characteristics;    -   (x) modified growth or reproductive characteristics; and    -   (xi) improved harvest, storage, or processing quality.

Suppression of the at least one endogenous miRNA decoy sequence isachieved by any means, including expression in the non-naturaltransgenic crop plant a gene suppression element (e.g., such as the DNAelements for suppressing expression described under the heading“Suppression of an endogenous or native miRNA”), or by any other meansof gene suppression.

In one non-limiting example, a transgenic plant overexpresses underconditions of nutrient sufficiency at least one miRNA decoy sequence fora miRNA that is natively expressed at high levels under conditions ofnutrient sufficiency and at low levels under conditions of nutrientdeficiency, thereby resulting in improved performance or yield undernutrient deficiency and improved nutrient utilization by the plant. Forexample, miRMON18 and miR399 are expressed at low levels duringnitrogen- or phosphate-deficient conditions, and at high levels undernitrogen- and phosphate-sufficient conditions, and thus their nativetarget genes are suppressed during nitrogen- or phosphate-deficientconditions and expressed at relatively higher levels under nitrogen- andphosphate-sufficient conditions; this results in improved nitrogenand/or phosphate utilization by the transgenic plant. Thus, a transgenicplant overexpressing a recombinant DNA construct including at least onemiRMON18 decoy sequence (or at least one miR399 decoy sequence) resultsin a higher level of expression of the miRMON18 native target genes (orof the miR399 native target genes) during nitrogen- andphosphate-sufficient conditions, relative to a plant in which therecombinant DNA construct is not expressed. In a non-limiting example, atransgenic plant overexpressing a recombinant DNA construct including atleast one miRMON18 decoy sequence is expected to accumulate relativelyhigher levels of the native miRMON18 targets (e.g., genes containing anSPX domain, such as the genes depicted in FIG. 12, as described inExamples 7, 9, and 10).

EXAMPLES Example 1

This example describes non-limiting embodiments of methods foridentifying crop plant (rice and maize) microRNAs and their precursor(foldback) structures, useful in making recombinant DNA constructs ofthis invention. Several small (19 to 25 nucleotide) RNA libraries werecloned from mature rice (Oryza sativa cv. Nipponbare) mature grain (3replicates) and seedling and from corn (maize, Zea mays) leaf and kernel(39 days after pollination) by high-throughput sequencing (Margulies etal. (2005) Nature, 437:376-380). The sequences thus obtained were usedfor miRNA prediction in rice genomic and maize genomic sequences,respectively, employing a set of rules derived from previouslycharacterized miRNAs, followed by manual inspection to eliminate poorlypredicted foldback structures. Small RNAs that matched perfectly toannotated tRNA, rRNA, transposon/retrotransposon and other knownrepeats, and chloroplast or mitochondria genomes were excluded from theanalysis.

The Institute for Genomic Research's rice genome annotation version 4.0(publicly available at www.tigr.org) was used to predict two flankinggenomic segments of ˜310 nucleotides in which a given small RNA waslocated near the left or right terminus of the segment (thus givingeither a sequence consisting of 280 nucleotides plus the small RNA plus10 nucleotides, or a sequence consisting of 10 nucleotides plus thesmall RNA plus 280 nucleotides. The foldback structure of each segmentthus obtained was predicted using the RNAfold program in the Viennapackage as described by Hofacker et al. (1994) Monatsh. f. Chemie,125:167-188. To facilitate the structure prediction, each small RNA wasassigned a pseudo-abundance of 2.

The structures were filtered based on characteristics of validated miRNAprecursors modified from those derived by Jones-Rhoades et al. (2006)Annu. Rev. Plant. Biol., 57:19-53. For rice miRNAs, the filteringrequirements included: (1) the small RNA must be located wholly withinone arm of the predicted foldback (stem-loop) structure; (2) the smallRNA and its counterpart segment in the opposite arm must have nucleotidesequences of at least 75% complementarity to each other; and (3) thesmall RNA and its counterpart, when forming the imperfect duplex, mustnot contain a symmetric bulge larger than 3 nucleotides or an asymmetricbulge larger than 2 nucleotides. The predicted structures satisfying theabove criteria were further filtered by selecting (1) only small RNAs oflength of 20 or 21 nucleotides and having a uracil as the 5′ terminalbase; or (2) the small RNA that were sequenced at least 10 times. Finalfiltering steps included: (1) selecting small RNAs with fewer than 23perfect matches to the genome to remove repetitive elements, and (2) thesegment used for the prediction could not include small RNAs from theminus strand. In cases where multiple overlapping small RNAs wereidentified, the most abundant member of the cluster was chosen as therepresentative sequence.

In the case of maize miRNA prediction, the prediction/filteringprocedures were modified from those used for the rice miRNAs, since acomplete maize genome is not yet available. Small RNAs from the maizeleaf and kernel libraries were analyzed independently to facilitate useof small RNA abundances for miRNA prediction. Small RNAs were mapped toMaize Assembled Gene Islands (MAGI version 4), a publicly available,assembled corn genomic sequence dataset as described by Fu et al.(2005), Proc. Natl. Acad. Sci. USA, 102:12282-12287. Sequences withsmall RNAs arising from both plus and minus strands were excluded.MicroRNA foldback structures were predicted and filtered using the samerequirements as for rice, and were further manually inspected toeliminate structures with large (>100 nucleotide) or highly unpairedloop regions. Previously characterized miRNAs excluded by filters wereused as an indicator of false negatives.

A total of 260676 unique small RNAs from rice in the size range of 19-25nucleotides were analyzed for putative novel miRNAs. After filtering andmanual inspection, 840 small RNAs corresponding to 1072 loci, wereidentified as novel rice miRNAs. Of the 27 known miRNA families presentin the miRNA database “miRBase” (available atmicrorna.sanger.ac.uk/sequences/) and in the original unique sequenceset 22 families were captured after filtering. The false negatives rateof 18.5% percent estimated based on characterized miRNAs (miRBase)indicate that the majority of miRNAs were captured by this approach.From a total of 126691 small RNAs from corn kernel, 116 novel maizemiRNAs corresponding to 281 loci in the MAGI version 4.0 corn genomicsequence were identified; similarly, from a total of 53103 small RNAsfrom corn leaf, 79 novel maize miRNAs corresponding to 302 loci wereidentified. The rice and maize miRNAs and their corresponding miRNAprecursor sequences, as well as the nucleotide position of the maturemiRNA in each miRNA precursor sequence, are referred to by theirrespective sequence identification number in Table 1 as follows: cornkernel miRNAs (SEQ ID NOS. 1-116), corn leaf miRNAs (SEQ ID NOS.117-195), rice miRNAs (SEQ ID NOS. 196-1035), corn kernel miRNAprecursor sequences (SEQ ID NOS. 1036-1316), corn leaf miRNA precursorsequences (SEQ ID NOS. 1317-1618), and rice miRNA precursor sequences(SEQ ID NOS. 1619-2690). The total of 174 predicted novel maize miRNAs(representing 528 genomic loci) included 9 miRNA orthologues that wereidentical to known miRNAs previously identified in species other thancorn; these are listed in Table 2.

TABLE 1 Maize and rice miRNAs and miRNA precursors miRNA pre-miRNANucleotide position of SEQ ID SEQ ID miRNA in pre-miRNA NO. NO. from to1 1067 11 31 1 1236 166 186 1 1269 166 186 2 1251 172 192 3 1115 167 1874 1240 11 31 5 1262 100 120 6 1074 11 31 6 1229 11 31 6 1234 11 31 61235 76 96 6 1274 11 31 6 1275 11 31 6 1276 11 31 6 1277 11 31 6 1278 1131 6 1279 76 96 6 1280 76 96 6 1281 11 31 6 1282 11 31 6 1283 11 31 61284 11 31 6 1285 76 96 6 1286 11 31 6 1287 11 31 6 1288 11 31 6 1289 1131 7 1205 70 90 7 1221 116 136 8 1041 11 31 8 1196 78 98 9 1110 92 113 91182 79 100 9 1255 11 32 10 1106 64 84 11 1194 64 84 12 1048 74 94 121257 11 31 12 1266 74 94 12 1267 74 94 13 1059 72 92 13 1068 11 31 131237 11 31 14 1226 11 31 14 1249 69 89 15 1066 11 31 15 1233 11 31 151256 11 31 15 1260 11 31 15 1265 115 135 15 1308 11 31 16 1129 128 14616 1199 11 29 17 1040 45 68 17 1246 11 34 17 1247 45 68 18 1131 104 12719 1119 142 162 19 1166 11 31 19 1169 143 163 19 1172 69 89 19 1175 1131 19 1177 143 163 19 1180 11 31 19 1186 11 31 20 1087 230 251 20 125811 32 21 1046 11 31 21 1157 63 83 21 1216 73 93 22 1254 102 122 23 126111 30 24 1125 70 90 24 1314 11 31 24 1315 11 31 25 1161 11 31 26 1124 1131 27 1198 77 96 28 1309 74 94 29 1114 11 29 29 1232 11 29 30 1192 70 9131 1077 72 92 32 1136 88 108 33 1054 38 58 34 1053 11 31 34 1096 11 3134 1292 84 104 34 1307 84 104 34 1313 85 105 35 1063 11 31 35 1214 11 3136 1156 11 31 37 1055 11 30 38 1291 83 103 39 1116 11 31 39 1138 11 3140 1201 82 102 41 1065 65 85 41 1070 11 31 41 1088 65 85 41 1113 11 3141 1154 11 31 41 1163 11 31 41 1173 63 83 42 1310 11 31 43 1122 105 12544 1159 86 106 45 1081 11 31 46 1104 11 31 47 1108 11 31 48 1057 11 3148 1162 185 205 49 1112 127 147 49 1130 11 31 49 1144 11 31 49 1145 1131 49 1168 11 31 49 1195 115 135 49 1211 122 142 49 1215 11 31 49 1217127 147 49 1219 11 31 50 1056 11 30 51 1036 60 80 51 1089 11 31 52 114364 84 53 1060 36 56 54 1058 206 226 54 1064 199 219 54 1128 199 219 541224 197 217 54 1242 200 220 54 1272 11 31 54 1312 11 31 55 1141 70 9056 1061 11 30 57 1183 114 134 58 1140 132 152 59 1126 11 31 60 1181 1131 61 1204 47 67 62 1037 89 109 62 1071 11 31 62 1146 11 31 62 1148 1131 62 1270 11 31 63 1227 11 31 63 1231 11 31 63 1243 11 31 63 1295 151171 63 1296 151 171 63 1297 151 171 63 1298 151 171 63 1299 11 31 631300 11 31 63 1301 151 171 63 1302 11 31 63 1303 11 31 63 1304 152 17263 1305 11 31 64 1038 101 120 64 1084 11 30 64 1127 11 30 64 1133 11 3064 1147 101 120 64 1160 11 30 64 1170 183 202 64 1171 11 30 64 1185 1130 64 1190 11 30 64 1193 11 30 64 1206 11 30 64 1208 11 30 64 1244 11 3064 1245 11 30 64 1253 11 30 64 1259 11 30 64 1268 183 202 65 1098 241261 65 1189 241 261 66 1045 11 31 66 1252 133 153 67 1094 71 91 68 115211 31 68 1158 11 31 68 1203 11 31 69 1097 43 63 70 1103 11 31 71 1239 4161 72 1044 11 31 73 1271 44 64 74 1042 76 96 75 1230 11 31 76 1149 11 3177 1218 11 30 78 1073 141 161 79 1047 11 31 80 1293 82 101 81 1080 11 3182 1316 11 31 83 1118 11 31 84 1050 11 31 85 1072 115 135 86 1085 31 5186 1241 31 51 87 1187 11 31 87 1197 11 31 87 1207 39 59 87 1213 38 58 881117 91 111 89 1101 47 67 90 1174 155 174 91 1209 149 169 91 1273 149169 92 1039 11 31 92 1228 55 75 92 1238 55 75 92 1250 55 75 93 1099 1130 94 1132 145 165 94 1139 140 160 94 1167 11 31 95 1052 11 31 96 104911 30 96 1105 11 30 97 1051 66 85 97 1100 136 155 98 1164 11 31 99 108673 93 99 1093 11 31 99 1294 70 90 100 1109 83 103 100 1111 11 31 1001137 11 31 100 1151 11 31 100 1179 36 56 100 1184 11 31 100 1210 11 31100 1222 72 92 101 1043 35 55 102 1311 67 87 103 1082 11 31 103 1120 1131 103 1165 147 167 103 1178 147 167 103 1220 11 31 104 1076 131 151 1041083 131 151 105 1102 11 31 105 1212 11 31 105 1225 11 31 106 1306 11 31107 1062 11 30 107 1075 11 30 107 1091 75 94 107 1121 11 30 107 1134 1130 107 1142 11 30 107 1176 75 94 107 1191 76 95 107 1200 76 95 107 124873 92 107 1263 76 95 108 1078 11 31 109 1135 75 95 110 1153 11 31 1111150 110 130 112 1123 11 30 113 1202 73 93 114 1223 144 164 115 1069 1131 115 1079 11 31 115 1092 11 31 115 1290 11 31 116 1090 11 31 116 1095182 202 116 1107 11 31 116 1155 193 213 116 1188 198 218 116 1264 199219 117 1366 11 31 117 1538 166 186 117 1578 166 186 118 1557 172 192119 1397 167 187 120 1449 213 233 120 1540 11 31 121 1572 100 120 1221507 189 208 123 1369 11 31 123 1534 11 31 123 1536 11 31 123 1537 76 96123 1583 11 31 123 1584 11 31 123 1585 11 31 123 1586 11 31 123 1587 1131 123 1588 11 31 123 1589 76 96 123 1590 76 96 123 1591 11 31 123 159211 31 123 1593 11 31 123 1594 11 31 123 1595 76 96 123 1596 11 31 1231597 11 31 123 1598 11 31 123 1599 11 31 124 1505 70 90 124 1522 116 136125 1324 11 31 125 1484 78 98 126 1466 79 100 126 1560 11 32 127 1389 6484 127 1401 11 31 128 1482 64 84 129 1337 74 94 129 1565 11 31 129 157674 94 129 1577 74 94 130 1559 11 31 131 1532 11 31 131 1554 69 89 1321365 11 31 132 1535 11 31 132 1562 11 31 132 1568 11 31 132 1575 115 135132 1612 11 31 133 1451 156 176 133 1519 107 127 134 1413 128 146 1341500 11 29 135 1406 232 252 136 1489 80 102 137 1543 11 31 138 1558 102122 139 1549 56 75 140 1571 11 30 141 1462 11 31 142 1514 11 31 143 161374 94 144 1611 68 88 145 1418 11 31 145 1459 11 31 145 1460 11 31 1461479 70 91 147 1320 11 31 147 1380 11 31 147 1381 11 31 147 1382 11 31147 1383 11 31 147 1384 11 31 147 1385 11 31 147 1409 97 117 147 1411100 120 147 1417 102 122 147 1422 11 31 147 1456 11 31 147 1483 11 31147 1493 100 120 147 1502 11 31 147 1517 100 120 147 1545 11 31 147 155011 31 147 1561 11 31 147 1564 11 31 147 1566 11 31 147 1569 11 31 1481372 72 92 149 1424 74 95 149 1616 11 32 150 1457 152 175 150 1513 152175 150 1515 152 175 151 1398 11 31 151 1442 11 31 151 1469 11 31 1511506 11 31 151 1520 191 211 151 1551 190 210 151 1581 192 212 152 150499 119 153 1503 82 102 154 1410 38 58 155 1487 200 220 156 1386 11 31156 1396 43 63 156 1546 11 31 156 1563 11 31 156 1601 11 31 157 1360 91110 157 1400 95 114 157 1415 95 114 157 1425 11 30 157 1426 92 111 1571453 11 30 157 1474 127 146 157 1480 56 75 157 1527 91 110 157 1552 1130 157 1570 57 76 157 1618 58 77 158 1394 11 31 159 1421 11 30 159 1450207 226 159 1495 11 30 160 1423 202 222 160 1529 11 31 160 1533 199 219161 1447 11 31 161 1555 11 31 162 1336 11 30 162 1579 11 30 163 1454 133153 164 1553 11 31 165 1343 11 31 165 1353 11 31 165 1468 168 188 1651475 113 133 165 1512 113 133 165 1547 107 127 166 1432 128 148 166 1548128 148 167 1350 11 30 168 1405 72 91 169 1429 38 58 170 1376 198 218170 1416 11 31 170 1440 11 31 170 1465 199 219 170 1614 11 31 171 1420138 158 172 1317 67 87 172 1326 67 87 172 1333 67 87 172 1338 67 87 1721339 68 88 172 1340 67 87 172 1341 67 87 172 1344 67 87 172 1346 68 88172 1348 67 87 172 1352 67 87 172 1431 68 88 172 1437 67 87 172 1448 6787 172 1458 68 88 172 1477 68 88 173 1582 36 56 174 1322 136 156 1741330 9 29 174 1349 11 31 174 1355 136 156 174 1399 137 157 174 1491 136156 174 1508 136 156 174 1509 11 31 175 1615 11 31 176 1392 117 136 1761438 43 62 176 1464 11 30 176 1490 11 30 176 1492 43 62 176 1498 11 30176 1499 43 62 176 1510 11 30 177 1371 11 31 178 1358 75 95 178 1364 5979 178 1390 11 31 178 1393 75 95 178 1395 11 31 178 1408 71 91 178 142857 77 178 1434 11 31 178 1436 75 95 178 1443 75 95 178 1455 56 76 1781461 75 95 178 1463 75 95 178 1467 75 95 178 1470 75 95 178 1476 11 31178 1485 11 31 178 1488 11 31 178 1496 11 31 178 1516 11 31 178 1518 1131 178 1523 75 95 178 1525 75 95 178 1528 11 31 178 1531 10 30 178 154211 31 178 1544 11 31 178 1556 11 31 178 1567 11 31 178 1573 11 31 1781574 75 95 179 1435 55 75 180 1402 113 133 180 1441 11 31 180 1521 103123 180 1617 105 125 181 1329 33 53 182 1334 11 31 182 1345 211 231 1821347 11 31 183 1452 36 56 184 1407 144 164 185 1404 11 31 185 1412 11 31185 1419 11 31 185 1481 11 31 185 1494 11 31 185 1524 11 31 185 1526 1131 186 1478 11 31 186 1511 11 31 186 1539 11 31 187 1430 112 132 1871439 241 261 188 1342 11 31 188 1541 11 31 189 1367 11 31 189 1391 63 83189 1414 11 31 189 1427 11 31 189 1530 11 31 189 1602 11 31 189 1603 1131 189 1604 11 31 189 1605 10 30 189 1606 11 31 189 1607 11 31 189 160811 31 189 1609 11 31 189 1610 11 31 190 1403 11 31 190 1471 11 31 1901497 11 31 191 1318 72 92 191 1319 72 92 191 1321 11 31 191 1325 11 31191 1327 11 31 191 1328 11 31 191 1331 72 92 191 1332 72 92 191 1335 7292 191 1351 11 31 191 1357 11 31 191 1359 75 95 191 1361 72 92 191 136211 31 191 1363 72 92 191 1368 11 31 191 1370 72 92 191 1373 72 92 1911374 72 92 191 1375 11 31 191 1377 11 31 191 1378 72 92 191 1379 72 92192 1356 11 31 193 1323 77 97 193 1354 11 31 193 1387 11 31 193 1388 1131 193 1433 112 132 193 1444 11 31 193 1445 11 31 193 1446 11 31 1931473 77 97 193 1486 11 31 193 1501 11 31 193 1580 75 95 194 1472 33 53195 1600 63 83 196 1663 281 299 197 2542 11 31 198 2532 11 31 199 197766 86 200 1946 11 30 201 2365 11 31 202 1735 34 53 203 2046 64 84 2041746 281 301 205 1778 11 31 206 2189 45 64 207 2549 11 31 208 2597 11 31209 2519 195 214 210 1829 257 277 211 2291 241 260 212 1938 11 30 2121994 11 30 213 2056 144 164 213 2265 137 157 214 1950 264 283 214 2039225 244 214 2148 225 244 214 2358 225 244 214 2491 209 228 215 2116 194213 216 2273 258 278 217 1631 252 270 218 1679 11 31 219 2304 11 30 2202071 85 105 221 1813 281 301 222 2604 201 220 223 2054 11 30 224 2653124 147 225 1761 134 153 226 2554 11 29 227 1713 259 277 228 2557 11 31229 1860 11 31 229 1922 41 61 230 2582 11 31 231 2309 104 122 232 191311 31 233 1747 11 31 234 1644 11 30 235 2174 11 31 236 2017 100 120 2362120 98 118 237 2010 113 132 238 2528 11 31 239 2417 11 30 240 1802 112130 240 2299 200 218 240 2591 243 261 240 2592 11 29 241 1643 11 30 2412122 11 30 241 2280 11 30 242 2489 11 30 243 2074 274 294 244 1890 266285 245 2139 193 216 246 1892 80 100 247 1861 55 77 248 2676 11 30 2492681 272 291 249 2682 11 30 250 2005 73 93 250 2092 73 93 250 2406 52 72251 2202 11 29 252 1919 239 258 253 2409 248 271 254 1926 11 31 255 2445247 267 256 1804 11 31 257 1774 149 168 258 2394 11 31 259 1697 87 107260 2268 37 55 261 2063 281 304 262 1686 11 31 263 2184 11 31 264 215848 67 264 2399 217 236 265 2353 11 34 266 2524 11 31 267 2183 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11 35 715 2064 11 31 716 2545 86105 717 1636 11 31 718 2248 11 30 719 2320 11 30 720 1739 99 119 7212286 11 31 722 2321 11 31 723 2216 36 55 724 1814 82 105 725 2288 11 30726 2256 245 263 727 1905 11 31 727 2440 11 31 728 1998 71 91 729 162454 74 730 1940 11 31 730 2328 111 131 731 1662 11 29 732 2118 11 30 7331809 60 79 733 2037 183 202 733 2150 73 92 734 2596 11 31 735 2414 11 31736 2192 11 31 737 2196 11 30 738 2521 39 59 739 1678 11 31 740 1791 152172 740 1853 158 178 740 2134 155 175 741 2058 129 149 742 1635 194 214742 1914 11 31 742 2362 11 31 743 1653 220 238 743 2104 281 299 744 230011 31 745 1648 82 102 746 2423 11 30 746 2648 11 30 747 2305 11 30 7481855 11 30 748 1961 11 30 749 1982 11 34 749 2022 11 34 749 2075 11 34749 2292 11 34 750 2344 11 29 751 2019 53 73 752 1758 74 94 753 1780 3252 754 2631 99 119 755 2564 11 31 756 2570 11 31 757 2574 44 63 758 188011 29 759 1799 11 31 760 2469 85 104 761 2066 11 30 762 1826 11 31 7632382 11 31 764 2132 233 253 764 2177 233 253 765 2688 11 31 766 1773 87105 767 2160 11 31 768 1945 225 244 769 2342 266 284 770 1712 102 121771 2087 11 30 771 2235 11 30 771 2567 11 30 772 2270 11 31 772 2312 1131 772 2383 11 31 772 2657 11 31 773 2176 178 197 774 1748 11 30 7741842 11 30 775 1723 45 64 775 2615 45 64 776 2072 11 31 777 1655 11 30778 1989 123 146 779 2032 11 30 779 2263 11 30 779 2425 11 30 779 262011 30 780 1805 11 30 780 2149 11 30 780 2331 11 30 780 2477 11 30 7802511 11 30 781 2563 164 184 782 2208 149 168 783 2186 275 294 784 182311 32 785 1879 11 31 786 2666 11 31 787 2490 279 298 788 1752 11 31 7892052 89 109 789 2628 89 109 790 1733 62 82 791 1777 11 31 791 2412 11 31791 2658 11 31 792 1898 281 301 793 1815 138 157 794 2456 181 201 7952112 159 178 796 1740 116 136 797 2359 11 32 798 2441 11 31 799 2585 1131 800 2665 11 31 801 2313 237 256 802 1941 11 30 803 1646 130 150 8042349 11 31 805 1720 147 167 805 2191 147 167 806 2100 11 30 807 1659 269289 808 2229 269 289 809 2443 281 300 810 2600 114 134 811 2215 216 236812 1693 11 30 813 1749 72 92 813 2156 72 92 813 2278 72 92 813 2416 7292 813 2551 116 136 814 2195 11 31 815 2281 271 291 816 2264 11 30 8171711 11 30 818 1981 11 31 819 1854 11 30 820 2413 148 168 821 2404 262282 822 2090 44 64 822 2654 112 132 823 2525 44 64 824 2402 128 147 8242606 135 154 825 2387 11 31 826 1762 11 31 827 1895 36 55 828 2467 11 31829 2102 11 31 830 2588 11 30 831 1671 11 31 832 1650 11 30 833 1843 281300 834 1976 30 49 834 2448 30 49 835 2125 11 30 836 2255 11 31 837 268337 56 838 2274 11 30 839 1634 11 31 840 2605 281 300 841 2497 124 143842 2685 11 31 843 2376 281 301 844 1925 82 102 845 2438 11 31 846 260711 31 847 2398 71 90 848 2459 165 185 849 2460 234 254 850 1975 11 30851 2579 11 30 852 2042 11 30 852 2385 85 104 853 1933 11 31 854 2159214 234 854 2369 214 234 855 2187 41 60 855 2237 44 63 856 2269 207 227857 2050 11 31 858 1987 82 101 859 1656 11 31 860 2210 11 31 861 1900 1130 861 1979 11 30 862 1727 44 64 863 2205 200 220 864 2164 53 71 8652498 11 30 866 1769 42 62 867 1726 31 50 868 1652 11 29 868 1730 156 174868 1884 11 29 868 1967 11 29 869 1821 279 299 870 1620 11 31 871 1767281 300 872 2155 11 31 873 1800 39 57 874 2501 11 31 875 2211 226 246876 2009 11 31 877 2110 11 31 878 1991 11 30 879 2044 11 29 880 1833 1131 881 2124 173 193 882 2408 11 31 883 1751 122 142 884 1744 11 31 8852289 190 209 886 1825 11 31 887 2171 11 30 888 2345 11 31 889 2034 50 69890 2059 11 31 891 2026 194 212 892 2573 11 32 893 1689 11 30 893 220111 30 894 1904 56 76 895 2209 11 30 896 1736 11 30 897 2295 100 120 8982330 11 31 899 1664 281 300 900 2641 54 74 901 2678 11 31 902 1932 11 31903 1841 11 31 903 2583 11 31 903 2661 11 31 904 2348 70 90 905 2650 1131 906 2507 46 65 907 1910 47 66 907 2618 35 54 908 2057 134 154 9092070 11 31 909 2667 11 31 910 2167 73 92 911 2310 68 88 912 2578 11 30913 1706 33 53 914 1670 34 54 915 1683 71 91 916 1754 11 31 916 2226 1131 917 2028 272 292 918 2214 11 30 919 1806 84 104 920 2333 281 301 9212316 11 30 922 2025 11 31 922 2315 11 31 922 2405 11 31 922 2437 11 31923 2357 261 281 924 2381 11 31 925 1871 82 101 926 2433 186 205 9272377 273 292 928 1632 270 290 929 2485 118 138 930 2540 11 30 930 260211 30 930 2670 11 30 931 1812 11 31 932 2294 240 260 933 2687 37 56 9342401 86 106 935 2084 11 31 936 1661 113 133 936 1939 112 132 936 2088112 132 936 2677 90 110 937 2078 11 30 938 1934 273 292 939 1851 280 298940 1817 11 30 941 1835 11 30 942 2495 11 30 943 2267 130 150 944 262111 31 945 1782 11 30 946 1669 11 31 947 2403 36 55 948 1927 11 31 9492287 11 31 950 2356 11 31 951 2015 131 151 952 2616 11 29 953 2486 64 83954 1899 255 275 955 1742 132 151 956 2493 11 30 957 1715 11 31 958 249211 31 959 1997 11 31 959 2213 11 31 960 1966 75 95 961 2012 11 31 9622224 11 31 963 2188 176 195 964 2598 58 78 965 2418 11 30 966 2444 11 30967 2372 11 31 968 1888 11 31 969 1651 11 31 970 2652 11 30 971 1965 275295 972 1743 11 31 973 1877 11 33 974 2386 274 293 975 2580 11 31 9762637 281 300 977 2577 39 59 978 1836 128 148 979 2452 11 30 980 2076 7696 981 1838 281 301 982 1690 11 31 983 2222 11 30 984 1935 11 31 9851816 11 30 986 1628 11 30 987 2504 11 31 988 2350 234 253 989 1831 11 30990 2065 11 30 991 2142 11 31 992 1896 11 31 993 1672 11 34 994 2624 1131 995 1959 97 117 996 2238 235 255 996 2612 271 291 997 1972 238 258998 2204 11 31 999 2496 94 114 1000 2055 79 99 1001 1691 11 34 1002 233611 30 1003 2096 11 31 1003 2539 131 151 1004 2085 11 31 1005 2298 11 311006 1641 245 264 1006 1996 11 30 1006 2203 11 30 1007 2679 153 173 10081741 11 31 1008 2322 11 31 1009 2642 11 29 1010 2442 11 31 1011 2575 1131 1012 2250 11 31 1013 1732 54 77 1013 2060 53 76 1013 2453 53 76 10142170 60 80 1015 2611 35 55 1016 2033 91 111 1017 2569 135 155 1018 236111 31 1019 1718 11 31 1020 2558 80 100 1021 2243 105 124 1022 2136 11 301023 1878 11 31 1024 2182 11 31 1025 1891 77 96 1026 1924 140 160 10272341 255 275 1028 2244 147 167 1028 2556 142 162 1028 2663 114 134 10291756 225 244 1029 2329 195 214 1030 1856 11 31 1031 1808 139 159 10321873 57 76 1033 1993 72 92 1034 1696 55 78 1035 1912 33 53 1035 1990 3353 1035 2500 33 53 1035 2664 33 53

TABLE 2 Maize miRNAs Predicted in Predicted in sRNA ID SEQ ID NO.Homolog* Corn Kernel Corn Leaf 15996 3 ptc-miR390c y y 19644 4osa-miR396e y y 25372 7 ptc-miR172f y y 35979 9 ath-miR167d y y 36116 10osa-miR528 y y 56811 133 ptc-miR396e n y 59250 16 ptc-miR398c y y 432006138 sbi-miR164c y y 1392730 32 ath-miR171a y n *“ptc”, Populustrichocarpa; “osa”, Oryza sativa; “ath”, Arabidopsis thaliana; “sbi”,Sorghum bicolor

Example 2

Rice genes predicted to be targets of the novel rice miRNAs werepredicted from The Institute for Genomic Research's rice genomeannotation version 4.0 (publicly available at www.tigr.org), based onsequence complementarity rules as described by Zhang (2005) NucleicAcids Res., 33:W701-704 and by Rhoades et al. (2002) Cell, 110:513-520.These predicted targets were sequences that included at least one miRNArecognition site recognized by a mature miRNA selected from SEQ ID NOS.1-1035, SEQ ID NOS. 2730-3921, SEQ ID NOS. 5498-6683, SEQ ID NOS.8409-8560, SEQ ID NO 8742, SEQ ID NO. 8744, SEQ ID NOS. 8812-8815, SEQID NO. 8845, and SEQ ID NO. 8850 or a mature miRNA derived from a plantmiRNA precursor sequence selected from SEQ ID NOS. 1036-2690, SEQ IDNOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ ID NO.8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819. Table 3 listsnon-limiting examples of miRNA recognition sites (SEQ ID NOS. 2691-2729)that are recognized by a rice mature miRNA (SEQ ID NO. 197).

TABLE 3Os_miRNA_60735 miRNA sequence UCCGUCCCAAAAUAUAGCCAC (SEQ ID NO. 197)miRNA recognition site Nucleo- No. of tide SEQ  mis-Predicted rice target position mRNA sequence ID match-Locus name and annotation  in locus corresponding to cDNA NO. Score esLOC_Os01g65130.1|11971.m126 1086-1106 guugcuauauuuugggacgga 2691 1 126|cDNA expressed protein LOC_Os11g37540.1|11981.m075 1925-1945guugcuauauuuugggacgga 2692 1 1 99|cDNA Serine/threonine-protein kinase Doa, putative,  expressed LOC_Os08g36030.1|11978.m0751126-1146 auugcuauauuuugggacgga 2693 1 2 92|cDNA Plant viral-responsefamily protein, expressed LOC_Os12g12470.2|11982.m268 1095-1115guuguuauauuuugggacgga 2694 1.5 2 68|cDNA NADP-dependentoxidoreductase P1, putative, expressed LOC_Os09g20410.1|11979.m052587-607 guugcuauauuuugggaugga 2695 1.5 2 48|cDNA hypothetical proteinLOC_Os06g12790.3|11976.m320 3792-3812 auuguuauauuuugggacgga 2696 1.5 328|cDNA RAC-like GTP binding protein ARAC10, putative, expressedLOC_Os11g24540.1|11981.m063 2004-2024 auuguuauauuuugggacgga 2697 1.5 380|cDNA signal peptide pepti- dase family protein, expressedLOC_Os02g38750.1|11972.m089 55-75 augcuuauauuuugggacgga 2698 1.5 363|cDNA hypothetical protein LOC_Os07g49480.2|11977.m290  2531-2551auuguuauauuuugggacgga 2699 1.5 3 28|cDNA expressed proteinLOC_Os01g56640.1|11971.m975 660-680 augauuauauuuugggacgga 2700 1.5 346|cDNA transcription factor jumonji, putative, expressedLOC_Os06g12790.2111976.m322  1947-1967 uuugcuauauuuugggaugga 2701 1.5 347|cDNA RAC-like GTP binding protein ARAC10, putative, expressedLOC_Os10g22560.1|11980.m052  1954-1974 uuugcuauauuuugggaugga 2702 1.5 363|cDNA POT family protein, expressed LOC_Os06g30280.1|11976.m075 1895-1915 guugcuauauuaugggacgga 2703 2 2 72|cDNA expressed proteinLOC_Os03g14800.3|11973.m069  1826-1846 aaagcuauauuuugggacgga 2704 2 317|cDNA aminotransferase, classes I and II family pro- tein, expressedLOC_Os05g25430.1|11975.m068 684-704 guuguuauauuuugggaugga 2705 2 336|cDNA protein kinase family protein, putative, expressedLOC_Os04g41229.2|11974.m789 175-195 guugcuauauuuuggcacgga 2706 2.5 264|cDNA Helix-loop-helix DNA- binding domain containingprotein, expressed LOC_Os01g06740.1|11971.m073  1362-1382gcaguuauauuuugggacgga 2707 2.5 3 04|cDNA Ribosome inactivatingprotein, expressed LOC_Os03g14800.2|11973.m351 861-881augcuuacauuuugggacgga 2708 2.5 4 26|cDNA aminotransferase,classes I and II family pro- tein, expressedLOC_Os09g36100.1|11979.m065  1469-1489 acaguuauauuuugggacgga 2709 2.5 460|cDNA expressed protein LOC_Os10g40740.1|11980.m068  1049-1069auacuuauauuuugggacgga 2710 2.5 4 42|cDNA Helix-loop-helix DNA-binding domain containing protein, expressedLOC_Os06g04970.1|11976.m052  989-1009 auacuuauauuuugggacgga 2711 2.5 426|cDNA expressed protein LOC_Os11g43760.1|11981.m082 2628-2648auacuuauauuuugggacgga 2712 2.5 4 11|cDNA Lipase family proteinLOC_Os12g11660.1|11982.m051 1479-1499 guugauuuauuuugggacgga 2713 3 345|cDNA expressed protein LOC_Os01g64330.1|11971.m425 630-650guugguuuauuuugggacgga 2714 3 3 82|cDNA expressed proteinLOC_Os07g04840.2|11977.m290 574-594 guugcuauauucuaggacgga 2715 3 360|cDNA Oxygen-evolving enhancer protein 2, chloroplastprecursor, putative, expressed LOC_Os09g13440.2|11979.m220 2068-2088auugcuauauuuuggaaugga 2716 3 4 62|cDNA expressed proteinLOC_Os06g12790.1|11976.m059 2271-2291 auugcuauauuuuggaaugga 2717 3 495|cDNA RAC-like GTP binding protein ARAC10, putative, expressedLOC_Os09g17730.1|11979.m050 958-978 uucccuauauuuagggacgga 2718 3 431|cDNA proton pump interactor, putative, expressedLOC_Os11g40390.1|11981.m078 1302-1322 augcuuauauuuuggaacgga 2719 3 485|cDNA expressed protein LOC_0s11g39670.1|11981.m078 924-944auugcuauauuauaggacgga 2720 3 4 14|cDNA seryl-tRNA synthetasefamily protein, expressed LOC_Os07g04840.1|11977.m049 4246-4266uugucuuuuuuuugggacgga 2721 3 4 51|cDNA Oxygen-evolvingenhancer protein 2, chloroplast precursor, putative, expressedLOC_Os03g47960.1|11973.m098 1125-1145 uuugcuauauuuugagaugga 2722 3 409|cDNA HECT-domain- containing protein, putative, expressedLOC_Os01g60780.1|11971.m122 3110-3130 ugcgauauauuuugggacgga 2723 3 406|cDNA integral membrane protein, putative, expressedLOC_Os10g01820.1|11980.m217 711-731 augcuuauauuuugagacgga 2724 3 447|cDNA expressed protein LOC_Os06g48030.3|11976.m321 1475-1495cuguauuuauuuugggacgga 2725 3 4 66|cDNA Peroxidase 16precursor, putative, expressed LOC_Os02g02980.1|11972.m056 1357-1377uuggcugaauuugggggcgga 2726 3 5 48|cDNA Enhanced diseasesusceptibility 5, putative, expressed LOC_Os08g32170.1|11978.m0721297-1317 uuggcugaauuugggggcgga 2727 3 5 11|cDNA oxidoreductase, 2OG-Fe oxygenase family protein, expressed LOC_Os11g39670.2|11981.m288114-134 guggcuuuauugugggguggu 2728 3 5 46|cDNA seryl-tRNA synthetasefamily protein, expressed LOC_Os08g25010.1|11978.m065 367-387cugguuaaauugugggaugga 2729 3 5 20|cDNA TBC domain containingprotein, expressed

Example 3

This example describes non-limiting embodiments of recombinant DNAconstruct wherein the at least one transcribable DNA element formodulating the expression of at least one target gene includes a DNAelement for suppressing expression of an endogenous miRNA derived from aplant miRNA precursor sequence selected from SEQ ID NOS. 1036-2690, SEQID NOS. 3922-5497, SEQ ID NOS. 6684-8408, SEQ ID NOS. 8561-8417, SEQ IDNO. 8743, SEQ ID NO. 8800, and SEQ ID NOS. 8816-8819. More specifically,this example illustrates non-limiting examples of DNA elements forsuppressing expression of a target gene, e.g., an endogenous miRNA or anendogenous miRNA decoy sequence.

FIG. 2A schematically depicts non-limiting examples of DNA elements forsuppressing expression of a target gene, e.g., an endogenous miRNA.These DNA elements include at least one first gene suppression element(“GSE” or “GSE1”) for suppressing at least one first target gene,wherein the first gene suppression element is embedded in an intronflanked on one or on both sides by non-protein-coding DNA. These DNAelements utilize an intron (in many embodiments, an intron derived froma 5′ untranslated region or an expression-enhancing intron is preferred)to deliver a gene suppression element without requiring the presence ofany protein-coding exons (coding sequence). The DNA elements canoptionally include at least one second gene suppression element (“GSE2”)for suppressing at least one second target gene, at least one geneexpression element (“GEE”) for expressing at least one gene of interest(which can be coding or non-coding sequence or both), or both. Inembodiments containing an optional gene expression element, the geneexpression element can be located outside of (e.g., adjacent to) theintron. In some embodiments, the intron containing the first genesuppression element is 3′ to a terminator.

To more clearly differentiate DNA elements of the invention (containingat least one gene suppression element embedded within a single intronflanked on one or on both sides by non-protein-coding DNA) from theprior art, FIG. 2B schematically depicts examples of prior artrecombinant DNA constructs. These constructs can contain a genesuppression element that is located adjacent to an intron flanked byprotein-coding sequence, or between two discrete introns (wherein thegene suppression element is not embedded in either of the two discreteintrons), or can include a gene expression element including a genesuppression element embedded within an intron which is flanked bymultiple exons (e.g., exons including the coding sequence of a protein).

FIG. 3 depicts various non-limiting examples of DNA elements forsuppressing expression of a target gene, e.g., an endogenous miRNA,useful in the recombinant DNA constructs of the invention. Where drawnas a single strand (FIGS. 3A through 3E), these are conventionallydepicted in 5′ to 3′ (left to right) transcriptional direction; thearrows indicate anti-sense sequence (arrowhead pointing to the left), orsense sequence (arrowhead pointing to the right). These DNA elements caninclude: DNA that includes at least one anti-sense DNA segment that isanti-sense to at least one segment of the at least one first targetgene, or DNA that includes multiple copies of at least one anti-senseDNA segment that is anti-sense to at least one segment of the at leastone first target gene (FIG. 3A); DNA that includes at least one senseDNA segment that is at least one segment of the at least one firsttarget gene, or DNA that includes multiple copies of at least one senseDNA segment that is at least one segment of the at least one firsttarget gene (FIG. 3B); DNA that transcribes to RNA for suppressing theat least one first target gene by forming double-stranded RNA andincludes at least one anti-sense DNA segment that is anti-sense to atleast one segment of the at least one target gene and at least one senseDNA segment that is at least one segment of the at least one firsttarget gene (FIG. 3C); DNA that transcribes to RNA for suppressing theat least one first target gene by forming a single double-stranded RNAand includes multiple serial anti-sense DNA segments that are anti-senseto at least one segment of the at least one first target gene andmultiple serial sense DNA segments that are at least one segment of theat least one first target gene (FIG. 3D); DNA that transcribes to RNAfor suppressing the at least one first target gene by forming multipledouble strands of RNA and includes multiple anti-sense DNA segments thatare anti-sense to at least one segment of the at least one first targetgene and multiple sense DNA segments that are at least one segment ofthe at least one first target gene, and wherein said multiple anti-senseDNA segments and the multiple sense DNA segments are arranged in aseries of inverted repeats (FIG. 3E); and DNA that includes nucleotidesderived from a miRNA, or DNA that includes nucleotides of a siRNA (FIG.3F).

FIG. 3F depicts various non-limiting arrangements of double-stranded RNAthat can be transcribed from embodiments of DNA elements for suppressingexpression of a target gene, e.g., an endogenous miRNA, useful in therecombinant DNA constructs of the invention. When such double-strandedRNA is formed, it can suppress one or more target genes, and can form asingle double-stranded RNA or multiple double strands of RNA, or asingle double-stranded RNA “stem” or multiple “stems”. Where multipledouble-stranded RNA “stems” are formed, they can be arranged in“hammerheads” or “cloverleaf” arrangements. In some embodiments, thedouble-stranded stems can form a “pseudoknot” arrangement (e.g., wherespacer or loop RNA of one double-stranded stem forms part of a seconddouble-stranded stem); see, for example, depictions of pseudoknotarchitectures in Staple and Butcher (2005) PLoS Biol., 3(6):e213. SpacerDNA (located between or adjacent to dsRNA regions) is optional butcommonly included and generally includes DNA that does not correspond tothe target gene (although in some embodiments can include sense oranti-sense DNA of the target gene). Spacer DNA can include sequence thattranscribes to single-stranded RNA or to at least partiallydouble-stranded RNA (such as in a “kissing stem-loop” arrangement), orto an RNA that assumes a secondary structure or three-dimensionalconfiguration (e.g., a large loop of antisense sequence of the targetgene or an aptamer) that confers on the transcript an additional desiredcharacteristic, such as increased stability, increased half-life invivo, or cell or tissue specificity.

Additional description of DNA elements and methods for suppressingexpression of a target gene can be found, for example, in U.S. PatentApplication Publication 2006/0200878, which is incorporated by referenceherein.

Example 4

This example describes non-limiting embodiments of methods for usingmicroRNAs, microRNA precursors, microRNA recognition sites, and microRNApromoters for modulating the expression of at least one target gene.

Various potential utilities of a miRNA or its recognition site arerevealed by the miRNA's expression pattern. Knowledge of the spatial ortemporal distribution or inducibility of a given mature miRNA'sexpression is useful, e.g., in designing recombinant constructs to beexpressed in a spatially or temporally or inducibly specific manner. Onenon-limiting method of determining a mature miRNA's expression patternis by isolation of the mature miRNA (or its precursor) and analyzing theexpression pattern by Northern blots with the appropriate probe (i.e.,probes specific for the mature miRNA or for the miRNA precursor).

FIG. 4 depicts a non-limiting example of Northern blot results formature miRNAs isolated from different maize tissues. One probehybridized to mature miRNAs from two families (miR156 and miR157).Individual mature miRNAs were expressed at differing levels in specificcells or tissues, e.g., Zm-miR390 was not expressed, or expressed onlyat low levels, in root and adult leaf, and miR156 is expressed in roots,leaves, and tassel. Thus, for example, recombinant DNA construct of thisinvention including a transgene transcription unit driven by aconstitutive promoter and a miRNA recognition site recognized by a maizemiR390 mature miRNA is useful for expression of the transgene in rootand adult leaf tissues but not in tissues where the mature miR390 isexpressed at high levels. To further illustrate use of the constructsand methods of the invention to control expression of a transgene, areporter gene is used as the transgene itself, or as a surrogate for thetransgene. For example, where expression of a reporter gene (e.g., greenfluorescent protein, GFP) is desired in maize stalk and immature eartissue, a miR156 target site is included in a GFP expression cassetteand expressed in a stably transgenic maize plant under the control ofthe CaMV 35S promoter. In tissues (e.g., roots, leaves, and tassel)where miR156 is strongly expressed, GFP expression is suppressed. Thesuppression phenotype may be limited to very specific cell types withinthe suppressed tissues, with neighboring cells showing expression or agradient of expression of GFP adjacent to those cells expressing themature miR156.

Another non-limiting method of determining a mature miRNA's expressionpattern is by analyzing transcription profiles of nucleic acid sequencesthat include the mature miRNA sequence, for example, by following ageneral procedure including the steps of:

(a) providing an initial miR sequence including the stem-loop region,e.g., from the publicly available miR sequences at the ‘miRBase”database (available on line at microrna.sanger.ac.uk/sequences);

(b) applying sequence analysis algorithms, such as BLAST as is wellknown in the art (see Altschul et al. (1990) J. Mol. Biol., 215:403-410)to identify homologous or identical sequences (e.g., from proprietarysequences on microarray probesets made with corn whole genome DNA); and

(c) analyzing the transcription profiles of the homologous probesetsequences identified in step (b) and identifying miRNAs having anexpression pattern in the desired tissues (i.e., male or femalereproductive tissues).

Preferably, a fourth step is added:

(d) for homologous probeset sequences found to have the desiredtranscription profiles, confirming identification of the miRNA gene byeither aligning the stem-loop sequence of the initial miR sequence tothe probeset sequence, or for potentially novel miRNAs, determining thesequence is predicted to fold into a stem-loop structure characteristicof a miRNA. Also preferably, an optional step is used, wherein one ormore BLAST comparisons against additional sequence datasets other thanthe probeset sequence dataset is included (prior to step (b) above),allowing the further identification of probes that fall outside of thepredicted fold-back region of the miR gene; false positives, e.g., dueto matches in the additional sequence dataset(s) that includeincorrectly spliced contigs, are identified by their lack of miRNAcharacteristics such as proper fold-back structure, and removed.

FIG. 5 depicts transcription profiles of probeset sequences that wereidentified, using the procedure described in the preceding paragraphs,as including miRNA precursor sequences having expression patternsspecific to maize male reproductive tissue (pollen). Such miRNAprecursors are suitable for use in recombinant DNA constructs of thisinvention designed for expression of a native miRNA (in this example, apollen-specific miRNA) under non-native conditions (e.g., under thecontrol of a promoter other than the promoter native to the miRNAprecursor). These miRNA precursors are also useful for providing a“scaffold” sequence that can be modified or engineered to suppress atarget gene other than the native or endogenous target gene. Onenon-limiting example of a recombinant DNA construct of this inventionincludes a strong constitutive promoter that is used to drive expressionof transgene transcription unit encoding a Bacillus thuringiensisinsecticidal protein or protein fragment (“Bt”), and a recognition sitefor a pollen-specific miRNA, resulting in strong Bt expression intissues of the plant except for the pollen. Additionally, the nativepromoters of these miRNA precursors are useful for pollen-specificexpression of any gene of interest.

In an alternative approach, an existing (native or endogenous) miRNArecognition site is identified, for example, using sequencecomplementarity rules as described by Zhang (2005) Nucleic Acids Res.,33:W701-704 and by Rhoades et al. (2002) Cell, 110:513-520. The nativemiRNA recognition site is mutated (e.g., by chemical mutagenesis)sufficiently to reduce or prevent cleavage (see Mallory et al. (2004)Curr. Biol., 14:1035-1046). In this way a gene containing a native miRNArecognition site and having desirable effects, e.g., increased leaf orseed size, can be mutated and thus expressed at levels higher than whenthe unmutated native or endogenous miRNA recognition site was present.One embodiment is to replace a native gene with an engineered homologue,wherein a native miRNA has been mutated or even deleted, that is lesssusceptible to cleavage by a given miRNA.

Another specific example of this approach is the inclusion of one ormore recognition site for a mature miRNA not substantially expressed inmaize roots but expressed in most other tissues (such as, but notlimited to, miRNA162, miRNA164, or miRNA390 as depicted in FIG. 4) in arecombinant DNA construct for the expression of a Bacillus thuringiensisinsecticidal protein or protein fragment (“Bt”, see, for example, the B.thuringiensis insecticidal sequences and methods of use thereofdisclosed in U.S. Pat. No. 6,953,835 and in U.S. Provisional PatentApplication No. 60/713,111, filed on 31 Aug. 2005, which areincorporated by reference herein) as the transgene, e.g., in a constructincluding the expression cassette e35S/Bt/hsp17. Including one or moreof these recognition sites within the expression cassette reduces theexpression of transcripts in most tissues other than root, but maintainshigh Bt target RNA expression levels in roots, such as is desirable forcontrol of pests such as corn rootworm. In similar embodiments,combinations of different miRNA recognition sites are included in theconstruct to achieve the desired expression pattern in one or morespecific tissues.

Example 5

This example describes additional non-limiting embodiments of crop plantmicroRNAs and their precursor (foldback) structures, useful in makingrecombinant DNA constructs of this invention. A total of 1327933 uniquesmall RNAs (20 to 24 nucleotides long) were obtained by high-throughputsequencing of 30 corn (maize) libraries (Margulies et al. (2005) Nature,437:376-380). The sequences obtained were used for predicting cornmicroRNAs and their precursor structures from maize genomic sequencesusing the procedures described above in Example 1. In total, 1192 smallRNAs in 1576 proprietary maize genomic sequences were predicted to benew miRNAs. The corn miRNAs and their corresponding miRNA precursors, aswell as the nucleotide position of the mature miRNA in each miRNAprecursor sequence, are referred to by their respective sequenceidentification numbers in Table 4 as follows: corn miRNAs (SEQ ID NOS.2730-3921) and corn miRNA precursor sequences (SEQ ID NOS. 3922-5497).

TABLE 4 Maize and rice miRNAs and miRNA precursors Nucleotide positionof miRNA pre-miRNA miRNA in SEQ ID SEQ ID pre-miRNA NO. NO. from to 27303928 84 104 2731 4099 92 113 2732 3935 11 31 2733 4093 11 31 2734 513411 32 2735 4864 188 211 2736 4123 11 30 2737 4108 4 27 2738 5217 11 332738 5277 11 33 2739 4328 71 90 2740 4635 42 62 2741 4591 11 34 27423925 11 34 2743 4036 11 34 2744 4586 11 32 2745 5245 37 57 2746 5417 3053 2747 4527 171 190 2748 5486 11 32 2749 4440 11 32 2749 4428 11 322750 5469 241 261 2751 5066 11 30 2752 5095 61 82 2753 4468 113 133 27544924 37 59 2755 5242 11 30 2756 5292 83 103 2757 3959 11 33 2758 5489150 171 2759 3929 11 34 2760 5153 11 30 2761 4251 11 34 2762 5361 35 552763 3995 11 34 2764 4448 11 30 2764 4473 11 30 2765 4784 11 30 27664478 63 83 2767 4477 11 34 2768 4275 11 33 2768 4223 11 33 2769 5084 1131 2769 5063 61 81 2770 3985 11 34 2771 5384 11 32 2772 4053 11 34 27724058 113 136 2772 4057 113 136 2772 4059 11 34 2772 4051 104 127 27724056 11 34 2773 4215 11 34 2774 4718 215 237 2775 5098 37 56 2776 501111 30 2777 5262 62 82 2778 5022 11 30 2779 5369 47 66 2780 5038 11 342781 3974 1 24 2782 4933 103 124 2783 4380 89 109 2784 4752 11 33 27854341 209 232 2786 5408 11 31 2786 5356 11 31 2787 5048 37 57 2788 4920153 174 2789 5366 35 55 2790 4159 203 222 2791 4798 44 67 2792 4530 1131 2793 5269 11 31 2794 4334 6 29 2795 5287 11 30 2796 5362 11 30 27965396 11 30 2796 5344 11 30 2796 5310 11 30 2796 5360 11 30 2797 5440 118138 2797 5456 118 138 2798 4962 70 93 2799 4522 11 34 2800 4286 11 342801 4299 11 30 2801 4235 11 30 2802 4103 11 32 2803 5136 37 58 28035208 37 58 2804 4894 11 31 2805 4413 11 30 2806 4807 11 34 2807 4844 426449 2808 4022 118 141 2809 5312 11 30 2810 4270 52 71 2811 4233 8 302811 4244 253 275 2811 4293 75 97 2812 4517 55 77 2813 4456 40 59 28144258 87 107 2815 5276 242 261 2816 4638 54 77 2816 4815 54 77 2817 437811 34 2818 5298 33 54 2819 4208 11 30 2820 4187 11 30 2820 4176 11 302821 5333 103 123 2822 4958 11 31 2823 4500 11 34 2824 4373 11 34 28254024 11 34 2826 5407 47 67 2826 5448 11 31 2827 5479 230 253 2828 420153 72 2829 4709 204 224 2830 4525 141 160 2831 4876 125 147 2832 4122 1131 2833 4060 109 132 2833 4055 11 34 2834 5302 74 94 2835 5308 60 792836 4238 75 95 2837 5119 11 32 2838 4873 83 103 2839 4947 11 30 28404791 231 253 2841 4567 11 33 2842 5463 11 33 2843 4957 42 62 2844 520538 61 2845 4239 65 85 2846 5059 149 169 2847 3964 79 102 2848 4516 48 712849 4121 11 32 2850 4526 72 95 2851 4659 11 30 2851 4670 11 30 28514668 11 30 2852 4206 616 636 2853 5331 443 465 2854 4726 11 34 2855 460611 34 2855 4729 11 34 2855 4565 11 34 2856 4684 11 33 2857 4660 11 342858 5284 92 115 2859 5354 60 83 2859 5395 61 84 2859 5336 11 34 28595394 11 34 2859 5430 11 34 2859 5449 11 34 2859 5355 11 34 2859 5444 226249 2860 5070 56 77 2860 5077 11 32 2861 4086 111 134 2861 4089 11 342862 4461 298 321 2863 4663 54 75 2864 4878 37 57 2865 4965 11 34 28664232 11 34 2867 4007 40 62 2868 4991 64 83 2869 5180 35 56 2870 4247 7192 2871 4179 11 34 2872 5470 35 56 2873 3983 65 88 2874 4263 11 31 28755204 64 83 2876 4364 11 30 2877 5359 218 238 2878 4291 11 34 2879 421711 34 2880 4921 11 30 2881 4075 11 34 2881 4067 11 34 2882 4508 11 342883 5120 11 33 2884 4276 34 53 2884 4318 34 53 2885 4598 11 32 28865445 72 91 2887 4045 38 60 2888 5473 11 34 2888 5324 11 34 2889 4030 1133 2889 5363 116 138 2889 4039 11 33 2889 4035 11 33 2889 4029 106 1282889 4031 11 33 2889 4032 11 33 2890 4174 11 34 2891 5271 35 58 28915109 36 59 2891 5163 35 58 2891 5159 35 58 2892 4578 11 34 2892 4774 1134 2893 3934 155 178 2893 3923 11 34 2894 4504 11 31 2895 5196 60 802896 4863 11 33 2897 3953 11 34 2897 3967 46 69 2897 3955 11 34 28973949 49 72 2898 5397 11 32 2898 5392 11 32 2899 4383 36 59 2900 5240 3659 2901 5005 58 80 2901 4880 58 80 2902 4750 11 32 2903 4279 11 34 29044230 229 248 2905 4955 39 59 2906 4736 51 70 2906 4605 51 70 2907 5379138 158 2908 5435 11 32 2909 5127 11 31 2910 5368 11 31 2911 4877 11 342912 5023 50 73 2913 4087 11 32 2914 5303 174 193 2915 5461 54 77 29164789 44 63 2917 3937 11 34 2918 4218 135 158 2919 5040 119 139 2920 514711 34 2921 5329 54 77 2922 4135 67 86 2923 4981 122 141 2924 5071 11 322925 4620 45 65 2926 4008 11 34 2927 3994 252 275 2928 4280 11 34 29294142 77 97 2930 4982 11 30 2931 4917 4 27 2932 4126 106 128 2933 3945202 222 2934 4269 11 32 2935 4483 154 177 2936 5393 46 69 2937 4319 186208 2938 4080 69 92 2939 4918 11 33 2940 4499 11 30 2941 4327 304 3232942 5146 121 144 2943 5106 208 228 2944 4680 11 30 2945 4209 11 34 29465453 132 151 2947 4166 137 157 2948 5386 11 32 2949 5199 11 32 2950 496944 63 2951 5033 179 199 2951 5091 180 200 2952 4063 11 34 2953 5232 138158 2954 5494 11 31 2955 4972 11 30 2956 5008 245 268 2957 4111 6 262958 4501 37 56 2959 5349 11 34 2960 3971 11 31 2960 3956 217 237 29603963 92 112 2961 4044 54 76 2962 5050 11 33 2963 4421 116 138 2964 4083200 219 2965 4566 11 32 2966 4193 11 34 2967 5376 62 85 2968 4787 31 502968 4759 31 50 2969 4333 53 72 2969 4385 53 72 2970 4391 11 32 29714537 11 30 2972 5317 133 153 2972 5318 136 156 2972 5446 62 82 2972 5380136 156 2972 5471 162 182 2972 5452 136 156 2972 5341 140 160 2972 546011 31 2972 5374 11 31 2972 5451 137 157 2973 4084 11 34 2974 4116 41 602975 5468 11 31 2976 4474 11 34 2977 4744 11 34 2978 4447 11 34 29794738 11 30 2980 4132 11 34 2981 4505 11 30 2982 5124 11 33 2983 4888 3558 2984 4558 11 34 2985 4449 41 60 2986 5410 116 136 2987 5030 11 342988 4767 11 30 2988 4633 11 30 2989 5288 35 56 2990 4101 245 266 29915178 11 32 2991 5198 8 29 2991 5275 11 32 2991 5185 11 32 2991 5160 1132 2992 4097 11 34 2993 4656 11 31 2994 4514 219 241 2994 4398 219 2412995 5437 11 34 2996 4117 39 58 2997 4446 11 31 2998 5421 6 27 2999 425911 30 3000 5423 11 31 3001 4948 101 121 3002 5268 11 31 3003 4285 11 343004 5351 11 32 3005 5477 49 69 3006 5237 11 34 3006 5219 11 34 30065238 11 34 3007 4246 11 31 3008 4466 41 62 3009 3977 11 34 3009 3979 3760 3009 3975 37 60 3009 3972 38 61 3010 5112 11 32 3011 4085 254 2773012 5003 313 336 3013 4625 11 30 3014 5459 108 131 3015 5143 85 1043016 5334 34 57 3017 5482 11 30 3018 5326 139 161 3019 3941 69 92 30193938 69 92 3019 3943 69 92 3020 4682 11 30 3021 4214 115 138 3022 469111 32 3022 4753 11 32 3023 4717 11 30 3024 5018 78 97 3025 4776 11 313026 4105 11 34 3027 4115 11 30 3027 4167 173 192 3028 4892 640 659 30294154 11 32 3030 4749 11 32 3030 4783 11 32 3031 4611 179 200 3032 472111 34 3033 5365 118 138 3034 4705 112 131 3034 4570 113 132 3034 4827104 123 3035 4714 42 63 3035 4829 11 32 3035 4733 43 64 3036 4617 11 333037 4847 126 146 3038 5130 11 31 3038 5223 11 31 3038 5156 11 31 30385248 11 31 3038 5183 11 31 3038 5278 11 31 3039 4314 60 80 3040 4502 113136 3041 5327 117 140 3042 4363 11 31 3042 4503 11 31 3042 4388 107 1273043 4370 39 59 3044 4151 138 161 3045 5170 11 30 3046 4061 11 34 30464073 11 34 3046 4070 11 34 3046 4065 11 34 3046 4062 102 125 3046 406411 34 3046 4076 11 34 3047 4546 11 33 3048 5311 118 137 3049 4204 11 313050 5370 11 34 3050 5357 49 72 3051 4671 11 32 3051 4716 11 32 30525346 102 123 3052 5465 102 123 3053 4409 59 80 3053 4467 59 80 3054 500159 82 3054 4853 60 83 3055 5429 39 61 3056 4518 6 26 3057 5062 11 343058 4207 155 174 3059 4492 11 34 3060 5224 50 73 3061 4047 97 120 30623984 1 24 3063 5385 50 73 3064 5090 58 77 3065 3940 11 30 3066 5353 3656 3067 5358 11 34 3068 4697 206 226 3068 4674 207 227 3068 4560 206 2263069 5258 11 34 3070 5188 44 67 3071 4054 43 66 3072 4542 38 60 30724742 38 60 3073 5260 11 30 3074 5434 213 234 3075 5381 11 30 3076 431294 117 3077 4874 11 34 3078 4930 89 108 3079 4139 11 34 3080 5174 94 1163081 5121 120 139 3081 5184 121 140 3082 4722 11 34 3083 4836 11 30 30845193 11 34 3085 5315 115 138 3086 4664 172 195 3087 5162 11 31 3088 491470 89 3089 4110 220 243 3090 4102 11 34 3091 5279 11 34 3092 4141 193216 3093 4869 11 34 3094 5135 11 30 3095 5041 56 79 3096 4553 42 63 30975020 37 58 3097 5017 37 58 3098 5442 174 194 3099 5382 74 94 3099 533011 31 3100 4495 11 34 3101 4253 75 96 3102 3944 41 64 3102 3942 11 343103 5073 58 77 3104 4137 142 163 3105 4066 11 31 3106 5028 70 90 31075267 105 128 3108 5169 11 33 3109 4543 165 184 3110 4702 11 31 3111 431311 34 3112 5132 140 159 3113 5404 4 24 3113 5398 4 24 3114 5116 11 343115 4818 11 31 3116 4026 48 71 3117 5141 136 159 3118 4420 11 30 31195013 11 30 3120 4336 11 30 3121 3999 34 53 3121 3998 34 53 3122 4631 3758 3123 4127 11 30 3124 4180 11 31 3125 4104 11 34 3125 4106 11 34 31265047 11 31 3126 4908 11 31 3127 5350 38 59 3127 5457 38 59 3128 5467 4161 3129 4602 11 31 3130 5348 70 90 3131 5034 11 31 3132 4773 43 62 31324614 51 70 3132 4687 51 70 3133 5026 8 28 3134 4376 11 33 3135 4748 6687 3136 4402 160 180 3137 4379 188 208 3138 4779 115 135 3139 5426 52 713140 5295 38 57 3141 5094 11 31 3142 5415 50 73 3143 4794 104 125 31445250 11 34 3145 4735 11 34 3146 4799 11 34 3146 4703 6 29 3146 4615 4568 3147 4686 205 224 3148 3980 70 90 3149 4109 221 244 3150 4610 57 763151 5167 176 196 3152 4149 11 34 3153 4384 1 20 3154 4899 143 164 31554405 11 31 3155 4462 11 31 3156 4990 200 220 3157 4497 74 96 3158 404111 34 3159 4646 46 67 3160 4623 385 404 3161 5202 11 30 3162 4970 11 333163 4788 204 227 3164 4937 11 30 3164 4837 11 30 3164 4893 11 30 31654694 11 32 3165 4792 11 32 3166 5388 71 90 3166 5387 71 90 3167 4443 3558 3168 4867 59 78 3169 4692 29 48 3170 4224 11 31 3171 4498 11 34 31725273 11 32 3173 4927 54 77 3174 5024 11 31 3175 5431 265 285 3176 431540 63 3177 4575 11 33 3178 4459 193 216 3178 4340 194 217 3179 4194 1134 3179 4152 99 122 3180 5436 92 111 3181 5427 35 54 3182 4033 39 613183 4643 223 243 3183 4609 223 243 3183 4808 223 243 3183 4802 181 2013184 5145 227 250 3185 5029 67 90 3186 4261 185 207 3187 5036 169 1883188 4146 11 33 3189 4549 11 31 3189 4576 11 31 3189 4548 11 31 31894594 11 31 3189 4732 4 24 3190 5015 11 34 3191 5046 64 84 3192 5280 1134 3193 5343 152 173 3193 5352 11 32 3193 5490 11 32 3194 4465 11 343195 5142 11 32 3196 5039 11 31 3196 5053 11 31 3197 4521 68 88 31984975 11 30 3199 4415 11 33 3200 5261 37 59 3200 5113 11 33 3201 5087 1134 3201 4866 122 145 3202 4834 11 31 3202 4651 11 31 3203 4513 217 2393204 4175 57 80 3205 4839 11 34 3206 5253 41 60 3207 4881 11 33 32084715 11 31 3209 4618 400 423 3210 4648 11 30 3211 4362 45 65 3212 5149226 248 3213 5286 81 102 3213 5300 81 102 3214 5074 11 34 3214 4849 1134 3215 4762 52 72 3216 5377 11 34 3217 4751 34 53 3218 4841 11 30 32195186 11 31 3220 4520 30 49 3221 3986 116 139 3222 5420 149 168 3222 5450149 168 3223 5282 35 57 3224 5319 104 124 3225 5100 35 56 3226 4250 1131 3226 4268 11 31 3227 3936 111 131 3228 4191 37 60 3229 4740 11 343230 4300 118 141 3231 4819 11 32 3231 4689 11 32 3231 4662 11 32 32324658 10 33 3233 5340 154 176 3234 4375 11 33 3235 5131 9 31 3236 4724139 159 3237 4256 52 74 3238 5051 34 55 3239 4453 32 55 3240 5372 11 303241 4444 135 154 3241 4365 11 30 3242 4731 150 169 3243 5213 11 31 32445314 11 31 3245 4854 11 30 3246 5320 187 207 3247 4938 99 122 3248 448911 32 3249 5474 11 30 3249 5371 11 30 3250 5002 11 30 3251 4699 34 573252 4153 11 31 3253 4130 11 34 3254 5138 11 33 3255 5222 50 72 32555200 50 72 3256 4107 11 31 3257 4858 75 94 3258 4826 11 33 3259 5042 4467 3260 4696 11 32 3261 4953 221 241 3262 4931 11 32 3263 3987 212 2353264 3968 96 119 3265 4629 71 90 3266 4600 11 31 3266 4564 207 227 32674419 45 67 3268 4964 41 60 3269 4950 153 172 3270 4801 33 52 3271 4048238 261 3272 5118 11 34 3272 5151 11 34 3272 5264 11 34 3273 4357 11 323274 4009 11 32 3275 5265 11 30 3275 5225 29 48 3275 5126 11 30 32755270 11 30 3275 5251 105 124 3275 5137 1 20 3276 4213 218 238 3277 407136 59 3278 4678 11 31 3279 4377 11 30 3280 4895 40 59 3280 5108 40 593280 4997 40 59 3280 4840 40 59 3280 5014 40 59 3280 4890 40 59 32804851 40 59 3281 4506 11 30 3282 4353 38 59 3282 4346 38 59 3283 4234 257277 3283 4168 323 343 3283 4220 245 265 3284 4273 11 33 3285 5243 166187 3286 4393 142 165 3287 4359 11 30 3288 5211 11 34 3288 5175 11 343288 5283 11 34 3289 4308 157 178 3290 3982 11 34 3291 4052 11 32 32924589 11 34 3293 4852 52 72 3294 4389 118 141 3295 4768 36 57 3296 481211 32 3297 3978 37 56 3298 4761 11 30 3298 4685 11 30 3299 4825 11 333299 4745 11 33 3299 4770 11 33 3300 5086 56 78 3301 4486 11 31 33024870 41 61 3302 5060 41 61 3303 4321 11 34 3304 4185 49 69 3304 4136 4969 3304 4287 49 69 3304 4322 49 69 3305 4588 11 34 3306 5236 11 34 33074143 11 30 3308 4302 11 30 3308 4240 11 30 3309 4366 11 32 3310 5472 5273 3311 4649 11 30 3312 4490 11 34 3313 4642 113 133 3314 4559 232 2523314 4701 232 252 3315 4216 11 31 3316 4339 37 59 3317 4423 138 157 33185241 55 78 3319 5255 34 54 3320 3927 40 60 3320 3930 40 60 3320 3926 4060 3320 3932 45 65 3320 3922 39 59 3320 3924 132 152 3320 3933 40 603321 4254 60 79 3322 5166 213 236 3323 4647 61 81 3323 4657 61 81 33234603 61 81 3324 5235 73 96 3325 4406 34 55 3326 5207 8 30 3327 4800 1132 3328 4942 11 32 3329 4796 11 34 3330 4998 35 56 3331 4741 240 2613332 5306 60 83 3333 4555 181 203 3334 4909 41 60 3335 5304 11 34 33355383 11 34 3336 5462 31 50 3337 4636 11 33 3337 4683 11 33 3338 5105 4063 3339 4552 100 122 3340 4078 39 62 3341 4835 11 31 3342 5187 75 963343 4679 158 181 3344 4865 11 32 3344 4862 11 32 3345 5274 11 34 33464936 11 33 3347 4352 11 31 3348 4221 11 31 3349 5072 11 31 3349 5083 1131 3350 4695 117 139 3351 5230 71 94 3352 4961 167 187 3353 5227 11 303354 4147 92 115 3355 4536 11 33 3356 5443 110 130 3357 5321 247 2663358 4196 28 47 3359 5487 35 56 3360 4227 11 30 3361 4488 119 139 33624001 107 130 3362 4003 8 31 3362 4012 105 128 3362 4011 112 135 33634926 136 155 3364 5433 11 34 3364 3961 11 34 3364 4005 11 34 3364 396011 34 3364 3947 151 174 3364 3976 11 34 3364 3966 106 129 3364 3973 1134 3364 3950 11 34 3364 3958 11 34 3364 3962 11 34 3364 3969 11 34 33643957 11 34 3364 4002 104 127 3364 3954 11 34 3364 3952 7 30 3364 3946 1134 3364 3948 11 34 3364 4006 11 34 3364 3970 11 34 3364 3965 11 34 33655161 50 71 3365 5165 50 71 3365 5173 50 71 3366 4460 11 30 3367 4690 1131 3368 4337 87 110 3369 4534 201 222 3370 4040 7 30 3371 5004 11 343372 4720 41 63 3372 4554 40 62 3372 4688 40 62 3373 4401 11 31 33734355 139 159 3374 4418 11 33 3374 4412 11 33 3375 4098 38 58 3376 537811 31 3377 4562 11 30 3378 4813 38 57 3379 4804 153 173 3379 4613 153173 3380 4088 11 34 3381 4644 229 252 3382 4708 11 31 3383 5206 11 303384 4301 36 59 3385 4471 11 31 3386 4951 11 30 3386 5097 11 30 33864904 11 30 3386 5107 11 30 3386 5007 11 30 3387 4298 204 223 3388 498441 64 3389 5027 11 31 3390 4082 51 73 3391 4640 165 184 3392 4427 11 313393 4411 11 30 3394 4855 45 68 3395 5220 11 32 3395 5272 11 32 33964335 11 32 3397 5476 215 236 3397 5390 215 236 3398 4442 87 107 33994838 11 30 3400 4222 37 58 3401 4435 11 34 3402 3990 11 34 3403 5176 200219 3404 5297 49 72 3405 4095 61 84 3406 4906 11 32 3407 4172 11 31 34084226 74 96 3409 5195 79 98 3410 4941 33 54 3411 4330 11 31 3412 4666 1130 3413 4316 72 95 3414 5168 33 53 3414 5210 33 53 3415 5484 37 60 34155491 47 70 3415 5401 37 60 3415 5455 37 60 3415 5458 37 60 3415 5402 3861 3416 4960 11 34 3416 4919 11 34 3416 5035 11 34 3416 5093 11 34 34175406 64 84 3418 4512 56 78 3419 4793 197 217 3420 4712 42 61 3420 467542 61 3421 5103 11 30 3421 4923 11 30 3421 5043 11 30 3422 4203 11 303423 4369 255 274 3424 5488 11 31 3425 4857 11 33 3425 4910 11 33 34264403 11 34 3427 4356 75 98 3428 4354 11 32 3429 4766 11 31 3429 4730 1131 3429 4760 11 31 3429 4561 11 31 3430 5244 11 30 3431 5294 11 34 34324985 74 93 3433 4000 11 34 3434 4574 11 34 3435 4231 11 34 3436 4439 215238 3437 5454 84 104 3437 5328 78 98 3438 5228 11 30 3439 5432 34 533440 5144 11 30 3441 4424 136 159 3442 5290 11 30 3443 5332 11 31 34444090 11 34 3445 4743 190 210 3446 4438 11 34 3447 4069 11 31 3447 407211 31 3448 4392 11 34 3449 4828 11 31 3450 3992 81 104 3451 4868 45 643452 4128 11 31 3453 4338 111 134 3454 4288 11 31 3455 4202 11 30 34565010 11 34 3457 4928 48 71 3458 4601 43 64 3458 4723 43 64 3458 4582 1132 3458 4786 11 32 3459 4929 117 136 3460 4074 11 34 3461 4183 11 303462 4450 126 149 3463 4805 39 62 3464 4898 11 30 3465 4949 76 97 34664769 32 51 3467 4833 43 66 3467 4550 43 66 3468 4988 50 73 3469 4414 4766 3470 5064 34 55 3471 4381 117 138 3472 4939 11 34 3473 5115 11 323474 5139 11 33 3475 4907 83 102 3476 4343 11 34 3476 4348 42 65 34774091 54 75 3478 4932 60 80 3479 4775 11 32 3480 4345 11 32 3481 4900 1131 3482 4155 11 34 3483 5226 287 309 3484 4283 45 64 3485 4935 245 2653486 5214 72 91 3487 5189 46 69 3488 4529 156 179 3489 4367 11 34 34904563 11 32 3491 4585 11 30 3492 4049 114 137 3493 4795 39 60 3494 398811 30 3495 4050 74 97 3496 5335 11 32 3497 4634 11 30 3498 4934 11 303499 4624 65 84 3500 4911 76 96 3501 5466 11 33 3502 5316 146 165 35034832 34 54 3503 4756 34 54 3504 4342 11 33 3505 4622 37 56 3506 5289 1130 3507 5464 130 153 3508 5054 11 30 3509 4480 11 34 3509 4351 11 343510 5325 11 33 3511 5475 11 31 3512 5305 11 33 3513 4307 63 86 35144290 37 59 3515 5281 11 33 3516 4544 114 137 3516 4739 214 237 3516 468132 55 3517 4332 79 102 3518 4747 52 74 3519 5391 11 32 3520 5192 11 303521 4249 40 62 3522 4816 303 326 3523 4150 11 31 3523 4296 11 31 35234118 11 31 3523 4320 11 31 3523 4294 11 31 3523 4225 11 31 3524 5447 122 3525 4612 11 31 3526 5057 87 106 3527 5122 11 30 3528 5291 133 1533529 3931 123 143 3530 5068 119 138 3531 4971 32 52 3531 4885 32 52 35314915 33 53 3532 5079 11 30 3533 5293 53 76 3534 4331 11 32 3535 4094 231254 3536 4368 11 33 3536 4484 11 33 3537 5342 142 161 3538 4482 45 683539 4886 127 146 3540 4015 86 109 3540 4018 11 34 3540 4017 85 108 35404025 85 108 3540 4016 85 108 3540 4014 11 34 3540 4023 85 108 3540 402185 108 3540 4020 85 108 3540 4027 11 34 3540 4028 11 34 3540 4013 85 1083540 4019 85 108 3541 5140 11 30 3542 4823 185 205 3543 4806 11 32 35444954 11 30 3545 4292 11 30 3546 4184 11 32 3547 4989 11 31 3548 4496 1134 3549 4311 50 69 3549 4112 11 30 3550 4568 11 30 3550 4540 11 30 35514822 78 99 3552 5389 11 31 3553 5285 227 246 3554 5493 11 32 3555 4140153 172 3556 5065 41 60 3557 5323 11 34 3558 4974 11 33 3559 4297 303324 3560 4510 11 34 3561 4781 139 162 3562 4242 223 245 3562 4178 223245 3562 4177 223 245 3562 4190 223 245 3562 4257 223 245 3563 3996 1131 3564 4081 69 92 3565 4579 60 79 3566 5099 136 155 3567 4665 238 2593568 5069 101 120 3569 5367 11 32 3570 4010 50 73 3571 5413 11 31 35724532 11 32 3573 4654 11 31 3574 4422 49 71 3575 5080 11 34 3576 4979 1131 3576 5067 11 31 3577 4856 11 32 3578 4966 38 61 3579 4400 48 67 35804437 11 31 3581 5123 39 62 3582 5215 11 34 3582 5155 40 63 3582 5182 4063 3582 5266 40 63 3583 4672 11 32 3584 5082 51 71 3584 4913 37 57 35854956 88 111 3586 5203 11 31 3587 4455 133 152 3588 4145 11 34 3588 432311 34 3589 5412 11 31 3590 4161 11 32 3590 4252 11 32 3591 5212 133 1533591 5179 133 153 3592 4946 113 135 3593 4632 226 249 3594 5441 94 1173595 4551 11 32 3596 5078 11 33 3597 4430 11 32 3598 4170 43 65 35994266 11 33 3599 4267 11 33 3599 4165 11 33 3599 4305 11 33 3600 4282 3353 3601 5150 68 87 3602 5403 81 103 3602 5492 81 103 3603 4493 373 3923604 4211 11 34 3605 5075 11 31 3606 4973 11 30 3607 4884 107 126 36085438 11 31 3608 5416 11 31 3609 4780 214 234 3610 4426 11 33 3611 434411 32 3612 4887 163 186 3613 4144 11 31 3614 4667 102 125 3615 5249 3760 3615 5218 36 59 3615 5129 36 59 3616 4725 11 32 3616 4719 11 32 36164538 11 32 3616 4765 11 32 3616 4547 11 32 3616 4661 11 32 3616 4809 1132 3616 4604 11 32 3616 4541 11 32 3616 4650 11 32 3617 4349 11 30 36185400 57 79 3619 4515 11 31 3620 5114 11 32 3621 4569 92 112 3622 4830143 162 3623 4810 35 56 3623 4764 198 219 3624 4616 99 118 3625 4487 111131 3626 4472 43 62 3627 5337 11 34 3628 5158 11 31 3629 5309 11 34 36305418 94 117 3630 5439 94 117 3631 5424 36 56 3632 5307 42 65 3633 424511 33 3634 4523 134 157 3634 4507 41 64 3635 4653 119 142 3636 5171 3858 3637 5322 88 107 3637 5347 85 104 3638 4469 11 31 3638 4371 11 313638 4404 108 128 3639 4875 11 32 3640 5133 67 90 3641 5092 11 34 36424587 11 30 3643 4329 78 100 3644 4896 50 71 3645 3991 7 29 3646 4347 5174 3647 4037 94 114 3647 4038 11 31 3647 4034 104 124 3648 5104 81 1013649 4188 58 81 3650 4711 162 183 3650 4831 162 183 3650 4652 162 1833651 4079 11 33 3652 4219 11 34 3653 4959 131 150 3654 4173 240 263 36554134 11 30 3656 4361 11 30 3657 4451 11 31 3658 4871 11 33 3659 4043 1134 3660 4387 11 32 3661 4425 41 61 3661 4457 42 62 3662 5409 56 75 36625497 62 81 3663 4129 11 31 3664 4746 11 33 3665 5425 11 33 3666 4860 1131 3667 4655 11 32 3668 4967 11 30 3668 4843 71 90 3669 5485 11 30 36695338 11 30 3669 5478 11 30 3670 3989 11 33 3671 4978 11 30 3672 4325 1134 3672 4197 201 224 3672 4181 11 34 3673 4046 128 151 3674 5246 209 2323675 4952 34 53 3676 4976 62 83 3677 4163 11 30 3678 4445 11 30 36794777 84 103 3680 5422 47 70 3681 4524 42 61 3682 4068 11 31 3683 4511183 206 3684 5496 11 31 3685 4557 155 178 3686 4310 11 31 3687 5254 4668 3688 5000 44 64 3688 4905 11 31 3689 4848 150 170 3689 4903 150 1703689 4889 177 197 3689 4994 150 170 3690 4995 43 63 3690 4943 43 63 36915399 38 57 3692 5480 128 151 3693 4817 11 34 3694 5301 11 34 3695 500979 100 3696 4125 43 63 3697 4360 11 32 3698 4113 71 90 3699 4901 11 313699 4922 11 31 3700 5012 231 250 3700 4980 231 250 3701 4284 73 95 37024639 11 34 3703 4189 36 59 3704 5483 68 87 3705 5058 11 34 3706 5037 5777 3707 4382 31 51 3708 5364 11 31 3708 5419 11 31 3709 4669 11 32 37104519 151 174 3711 4693 11 30 3712 4433 11 34 3713 4790 11 33 3714 459567 89 3715 4248 41 60 3716 4164 156 176 3717 4993 110 133 3718 4186 1132 3718 4133 63 84 3719 4463 11 34 3720 4963 11 34 3721 4324 11 30 37214281 11 30 3722 4198 11 32 3723 4811 149 172 3724 4676 220 239 3725 470643 64 3726 4004 11 31 3727 5252 11 32 3728 5259 11 34 3729 5256 35 543730 4539 138 159 3730 4599 11 32 3731 4700 40 63 3732 4533 11 34 37334264 11 31 3734 4996 11 31 3735 4148 11 33 3736 4481 11 30 3737 4607 1130 3738 4627 242 261 3739 5239 37 58 3740 5154 11 34 3741 4925 11 323742 4754 101 120 3743 4785 70 89 3743 4619 70 89 3743 4597 70 89 37434797 105 124 3744 5191 11 34 3744 5172 11 34 3745 5177 176 199 3746 419211 32 3747 4326 11 31 3748 3939 11 34 3749 4983 11 34 3749 4944 11 343750 5101 94 113 3751 4374 69 92 3752 4987 11 30 3753 4572 11 30 37544755 43 66 3755 5234 11 34 3756 4396 11 30 3757 4120 212 231 3758 4210136 157 3759 5157 11 34 3760 3993 33 54 3761 5373 118 138 3762 4621 3756 3762 4581 37 56 3762 4803 37 56 3763 5052 5 24 3764 4372 11 31 37654429 11 31 3766 5375 227 250 3767 4883 44 64 3767 4916 44 64 3768 472811 34 3769 4757 45 67 3770 3951 11 31 3771 5164 41 64 3772 4556 11 323772 4821 162 183 3773 5428 44 67 3774 4491 11 31 3775 5263 116 136 37764205 11 30 3776 4236 32 51 3776 4243 230 249 3776 4195 33 52 3777 426052 75 3778 4727 51 74 3779 4160 11 31 3779 4157 11 31 3780 5117 79 993781 4434 162 181 3782 5021 11 34 3783 4479 11 30 3783 4395 11 30 37845411 181 203 3784 5414 214 236 3784 5339 214 236 3785 5209 134 154 37864265 11 30 3787 5128 11 30 3788 5181 86 105 3789 5194 11 30 3790 4778194 215 3791 4902 32 53 3791 4861 32 53 3792 4272 11 34 3793 5233 11 323794 4608 199 222 3795 4228 42 65 3796 4350 11 34 3797 5111 11 32 37984306 66 85 3799 5313 31 51 3799 5345 32 52 3800 5049 110 130 3801 523199 121 3802 5257 11 34 3803 4992 119 139 3804 4590 11 30 3805 4470 49 703806 5495 78 100 3807 5405 203 226 3808 4454 37 57 3808 4535 37 57 38095481 42 64 3810 4100 135 154 3811 4077 49 72 3812 4859 11 31 3812 508811 31 3813 5089 11 34 3814 4452 66 87 3815 4698 45 65 3816 4394 11 303817 4410 49 72 3818 4158 71 90 3819 4891 45 65 3820 4758 68 91 38214509 11 31 3822 4295 33 52 3822 4317 33 52 3823 5201 11 34 3824 5045 112131 3825 4237 79 102 3826 4897 11 30 3827 4485 233 254 3828 4408 28 513829 4417 11 32 3829 4528 36 57 3830 4303 179 202 3831 3981 11 34 38325076 11 31 3833 4580 11 32 3834 4494 11 31 3835 4119 11 34 3836 4637 92115 3837 4407 11 34 3838 4912 11 30 3839 4673 11 32 3840 4882 241 2613841 4278 76 96 3842 4879 11 32 3843 5125 29 48 3844 4476 41 64 38454641 11 31 3846 5216 11 33 3847 4241 49 70 3848 4945 11 30 3849 4162 1130 3850 5032 11 33 3851 4593 11 34 3852 3997 11 34 3853 4431 11 31 38544571 40 59 3854 4630 40 59 3855 4096 36 57 3855 4092 11 32 3856 5296 3859 3857 4626 11 30 3858 4212 104 127 3859 4577 177 197 3860 4229 11 303860 4182 11 30 3861 4968 11 30 3862 5299 11 32 3863 4042 11 34 38644475 11 32 3865 4386 34 54 3865 4432 34 54 3866 4441 11 32 3866 4416 1132 3867 4545 11 30 3868 4820 34 54 3869 5056 61 80 3870 5102 11 31 38715019 93 114 3872 4262 43 66 3872 4200 43 66 3873 5081 34 53 3873 5044 3453 3874 4986 122 141 3875 4271 11 31 3876 4397 43 62 3877 4850 104 1243878 4584 11 30 3879 5016 49 72 3880 4156 8 31 3881 4274 44 63 3881 413144 63 3881 4114 44 63 3881 4124 11 30 3881 4289 44 63 3882 5148 11 343882 5229 11 34 3883 4436 11 32 3884 4940 47 68 3884 4845 47 68 38845025 47 68 3885 5055 11 34 3886 4255 65 85 3887 5096 11 32 3888 4304 141161 3888 4277 141 161 3889 4358 11 30 3890 4645 11 34 3891 4977 11 303892 4628 11 32 3893 4592 37 57 3893 4772 37 57 3894 4458 11 32 38944464 11 32 3895 5006 11 34 3896 4399 11 30 3897 4999 46 65 3898 5110 5779 3899 5152 54 75 3900 4710 41 62 3901 4199 42 61 3902 4872 74 97 39034782 36 56 3904 4771 102 121 3905 4573 11 30 3905 4824 11 30 3905 471311 30 3905 4734 11 30 3905 4596 11 30 3905 4707 11 30 3905 4583 11 303906 4842 11 30 3906 5085 11 30 3907 5061 11 31 3908 5197 38 58 39094846 79 99 3910 4169 11 33 3911 5190 11 32 3912 4704 11 33 3912 4763 1133 3913 4531 37 56 3914 4737 11 32 3915 4814 11 33 3916 5221 132 1533916 5247 132 153 3917 4390 33 53 3918 4138 173 196 3919 4677 42 64 39204171 11 34 3920 4309 11 34 3921 5031 64 87

Example 5

This example describes additional non-limiting embodiments of crop plantmicroRNAs and their precursor (foldback) structures, useful in makingrecombinant DNA constructs of this invention.

Small-RNA libraries were prepared from maize (corn, Zea mays) or fromsoybean (Glycine max) grown under water stress and control conditions(Table 5). Drought stages for soybean were assessed using a relativescoring system from 1.0 (no effect or control) to 4.0; examples ofsoybean plants at each stage are illustrated in FIG. 6. The small RNAsequences thus obtained were used for predicting additional novelmicroRNAs and their precursor (foldback) structures from maize orsoybean genomic sequences, respectively, using the procedures describedabove in Example 1. From maize, 1186 maize miRNAs were predicted in 1725maize genomic sequences, and from soybean, 134 soybean miRNAs arepredicted in 181 soybean genomic sequences (Table 6). These miRNAs andtheir corresponding miRNA precursor sequences, as well as the nucleotideposition of the mature miRNA in each miRNA precursor sequence, arereferred to by their respective sequence identification numbers in Table6 as follows: maize miRNAs (SEQ ID NOS. 5498-6683), corn miRNA precursorsequences (SEQ ID NOS. 6684-8408), soybean miRNAs (SEQ ID NOS.8409-8560), and soybean miRNA precursor sequences (SEQ ID NOS.8561-8417).

TABLE 5 Develop- Library mental Number Crop Plant Tissue stage Treatment42 maize (Zea mays) young sink leaf V8 control 43 maize (Zea mays) youngsink leaf V8 mild drought 44 maize (Zea mays) young sink leaf V8 control45 maize (Zea mays) young sink leaf V8 severe drought 46 maize (Zeamays) root V8 control 47 maize (Zea mays) root V8 mild drought 48 maize(Zea mays) root V8 control 38 soybean (Glycine seedling leaf seedlingcontrol max) 39 soybean (Glycine pooled seedling seedling drought, stagemax) leaf 3.0 and 3.5 pooled * 40 soybean (Glycine root mature controlmax) 41 soybean (Glycine pooled root mature drought, all max) maturestages 1.5 through 3.5 pooled * * For libraries 39 and 41 prepared fromsoybean, samples from the stages indicated were pooled; drought stagesfor soybean are assessed using a relative scoring system as follows: 1.0= no effect 1.5 = meristem or one trifoliate wilted 2.0 = twotrifoliates wilted 2.5 = all trifoliates wilted 3.0 = bottom trifoliatecompletely dried out and brittle 3.5 = all trifoliates but the top onecompletely dried out and brittle, top trifoliate still soft 4.0 = allcompletely dried out and brittle

TABLE 6 Maize and soybean miRNAs and miRNA precursors Nucleotideposition of miRNA pre-miRNA miRNA in SEQ ID SEQ ID pre-miRNA NO. NO.from to 5498 8176 11 34 5499 7089 11 34 5500 6978 99 119 5500 8029 97117 5500 8030 97 117 5500 8195 99 119 5500 8205 99 119 5500 8236 99 1195501 7554 143 164 5502 7902 11 34 5503 6723 57 80 5503 7359 11 34 55047087 11 34 5504 8156 11 34 5505 7303 62 85 5506 8120 35 56 5507 8047 1132 5508 6970 5 28 5509 7685 74 97 5510 6881 63 84 5511 7068 70 91 55118217 69 90 5512 7772 55 74 5513 7585 11 34 5514 7009 42 61 5514 7335 4261 5514 7912 42 61 5514 8272 42 61 5514 8354 42 61 5515 8130 11 32 55167085 55 77 5517 7845 11 32 5518 8270 135 158 5519 7363 50 69 5520 785311 32 5520 8147 11 32 5521 7710 11 30 5522 7521 2 25 5522 7655 187 2105523 7152 11 34 5524 6774 145 167 5525 6790 159 179 5526 7947 11 34 55277744 6 25 5528 7233 11 32 5529 7476 11 30 5530 7933 11 33 5531 7971 1134 5532 6815 11 34 5533 8034 41 63 5534 6824 11 31 5535 6912 136 1555535 7220 137 156 5535 7779 140 159 5536 7920 45 65 5537 7921 11 35 55388284 11 31 5539 6829 143 164 5539 6864 11 32 5539 6905 11 32 5539 6952144 165 5539 7709 11 32 5540 7045 91 115 5540 7207 91 115 5540 7641 91115 5540 7834 91 115 5540 7835 91 115 5541 8303 11 31 5542 8230 109 1325543 7839 34 55 5543 7840 34 55 5544 6856 11 34 5545 6686 204 224 55457092 199 219 5546 7888 38 61 5547 7293 11 34 5548 7871 11 32 5549 797939 62 5550 7324 11 34 5550 7922 157 180 5551 7691 45 68 5552 8233 40 635553 7170 47 70 5554 8390 36 59 5555 7177 11 31 5556 7525 61 81 55576991 39 62 5558 7879 111 132 5559 7627 11 32 5560 7485 11 34 5560 756540 63 5560 8056 11 34 5561 6690 11 34 5562 6832 54 75 5563 7459 45 655563 7460 45 65 5563 7507 46 66 5563 7730 45 65 5563 7756 46 66 55638058 46 66 5563 8064 47 67 5563 8065 46 66 5564 7571 47 70 5565 7030 1130 5566 7059 11 34 5566 7514 11 34 5566 8111 11 34 5566 8215 11 34 55668337 11 34 5567 6748 66 89 5568 6725 97 117 5568 6830 97 117 5568 830711 31 5569 7287 11 31 5570 6778 11 31 5570 8172 213 233 5571 7882 45 665572 7227 56 79 5573 7491 33 56 5574 7296 122 142 5575 7614 11 34 55766793 112 132 5577 6741 40 63 5577 6942 39 62 5577 7057 40 63 5577 720639 62 5577 7381 40 63 5577 7388 39 62 5577 7423 40 63 5577 7427 39 625577 7504 40 63 5577 7907 39 62 5577 7956 40 63 5577 7958 39 62 55777965 39 62 5577 7966 40 63 5577 7997 39 62 5577 8011 40 63 5577 8025 4063 5577 8170 39 62 5577 8185 39 62 5577 8312 40 63 5578 7978 58 81 55788131 58 81 5579 7430 62 83 5579 7442 62 83 5579 7698 62 83 5580 7914 5578 5581 6869 113 136 5582 7396 11 34 5583 7668 11 31 5584 6762 80 1035585 7937 11 30 5586 8175 11 31 5587 7477 73 96 5588 8180 37 60 55897934 35 58 5590 7517 123 146 5591 7566 11 34 5592 7781 11 32 5593 776087 110 5594 7223 11 31 5594 7417 11 31 5594 7496 11 31 5594 7816 11 315594 7872 11 31 5594 7897 11 31 5594 8024 11 31 5594 8026 11 31 55956958 40 63 5595 8016 11 34 5596 8285 76 99 5597 8125 11 34 5598 8008 4263 5599 7414 38 61 5600 6708 74 93 5600 8041 11 30 5600 8042 11 30 56017823 11 34 5602 7483 338 359 5603 6685 71 91 5603 7780 102 122 5604 7072118 138 5605 7893 11 31 5606 7646 70 92 5607 8348 104 127 5608 7290 6080 5609 7670 189 212 5610 6949 92 112 5611 6870 39 61 5611 6878 218 2405611 7243 218 240 5611 8235 39 61 5612 7546 11 32 5613 8264 11 34 56147829 11 31 5614 8314 11 31 5615 6760 11 31 5616 7310 51 71 5617 7373 1132 5618 7440 11 34 5619 7475 211 234 5620 8356 404 423 5621 7715 11 325622 6993 99 122 5623 7841 11 31 5624 7183 81 101 5625 6861 35 55 56268122 11 34 5627 6767 11 33 5627 7919 11 33 5628 6957 11 34 5629 7383 7395 5629 7634 73 95 5630 7809 38 59 5630 7810 37 58 5630 7936 38 59 56317140 40 61 5631 7197 40 61 5632 6730 215 234 5632 7754 218 237 5633 7577134 153 5633 7613 134 153 5634 7003 72 91 5635 7812 11 30 5636 8049 6083 5637 8375 11 33 5638 8164 11 31 5639 7573 37 56 5640 8259 11 34 56417240 11 30 5642 8128 222 245 5643 7660 11 32 5644 6804 37 59 5644 692437 59 5644 7138 37 59 5644 7439 37 59 5644 7450 37 59 5644 7588 37 595644 7591 37 59 5644 7786 37 59 5644 7905 37 59 5644 7928 37 59 56458204 38 60 5646 6687 187 209 5647 7330 11 32 5648 7587 11 34 5649 7435292 315 5650 7653 11 34 5650 8066 11 34 5651 7055 11 33 5651 7763 11 335652 7755 11 34 5652 7828 11 34 5653 8115 32 51 5654 7821 48 71 56556694 11 33 5656 6990 11 31 5656 7793 11 31 5657 8250 11 30 5658 8080 1134 5659 7394 11 34 5660 8305 11 31 5661 6873 11 31 5662 7264 51 74 56628287 11 34 5663 7001 11 32 5664 8394 11 34 5665 7406 78 97 5665 7470 1130 5666 7144 41 62 5666 7635 41 62 5667 6698 11 34 5668 7117 11 32 56687154 11 32 5668 7457 11 32 5669 8135 117 137 5670 6906 58 78 5671 716711 34 5672 7217 165 184 5672 7326 165 184 5673 6977 66 85 5674 7526 130149 5675 7820 11 32 5676 7765 11 34 5677 7110 48 69 5678 7319 11 35 56798171 46 65 5679 8241 46 65 5680 7555 31 54 5680 8143 31 54 5681 6707 1131 5682 8129 1 22 5683 7060 11 31 5683 7078 52 72 5684 7802 11 34 56857356 11 34 5686 6847 11 34 5687 8326 11 31 5688 7314 144 164 5689 751211 34 5690 8193 157 177 5691 8210 11 34 5692 7323 47 69 5693 8278 11 345694 8393 69 89 5695 8004 70 91 5696 6765 36 55 5696 7556 36 55 56967771 36 55 5697 7601 83 104 5698 7266 11 35 5699 7873 137 157 5700 709311 34 5701 7034 11 30 5701 7246 11 30 5702 6968 56 79 5703 7469 42 635704 7891 11 34 5705 8166 11 31 5706 7295 58 78 5707 7752 11 32 57086909 11 34 5708 7581 97 120 5709 8155 55 75 5710 7704 11 33 5711 8032 1131 5712 7868 11 34 5712 7869 11 34 5713 6766 42 62 5714 7644 11 34 57157132 75 98 5715 7245 11 34 5715 8276 11 34 5715 8344 11 34 5716 6781 1133 5717 6754 11 35 5718 7358 11 31 5718 8178 11 31 5719 7895 29 49 57207813 11 31 5721 7631 11 31 5722 7105 11 31 5723 7063 42 61 5723 7163 4261 5723 8308 42 61 5724 6734 11 32 5724 7121 11 32 5724 7632 11 32 57258046 11 32 5725 8396 11 32 5726 7836 211 232 5727 8148 11 34 5727 814949 72 5728 7175 11 33 5729 7134 11 34 5730 6858 86 108 5731 6836 11 335732 7910 107 126 5733 6884 233 256 5734 7010 11 30 5735 8158 11 30 57366989 11 33 5737 7964 11 34 5738 7041 76 97 5738 8268 75 96 5739 6850 1132 5740 6724 182 204 5740 8209 84 106 5741 7193 181 204 5741 7194 191214 5741 7798 181 204 5742 7354 83 104 5743 6716 50 73 5743 6736 11 345744 7253 240 262 5745 8084 97 120 5746 6853 113 133 5747 7564 11 315747 8330 11 31 5748 7166 11 34 5749 7182 96 116 5750 8222 211 231 57516860 178 199 5752 7018 11 34 5753 7026 11 31 5754 7484 124 145 5755 825511 31 5756 7944 11 34 5757 7297 11 34 5758 7650 11 30 5759 8174 46 675760 7474 11 34 5760 7510 11 34 5760 7511 11 34 5761 7360 11 30 57618246 11 30 5762 8403 29 52 5763 7005 64 86 5764 8310 11 33 5765 6894 324 5766 7558 46 67 5767 7520 232 252 5767 8017 148 168 5767 8100 70 905768 7611 236 255 5769 8345 107 127 5770 7025 11 31 5771 7143 11 34 57727447 11 32 5773 7590 11 34 5774 6739 152 175 5774 7669 151 174 5774 8013152 175 5775 6720 11 32 5775 7165 11 32 5775 7638 11 32 5775 8377 11 325776 7686 74 97 5777 7340 11 32 5777 7397 11 32 5777 8239 77 98 57788192 11 30 5779 8048 11 34 5780 7239 78 101 5781 7945 11 31 5782 8408 1131 5783 8057 11 30 5784 7204 237 256 5785 7599 135 158 5786 6921 11 315787 7677 11 31 5788 8124 55 76 5789 7817 11 32 5790 6934 11 32 57907015 11 32 5790 7124 11 32 5790 7317 11 32 5790 7428 11 32 5790 7488 1132 5790 7532 11 32 5790 7575 11 32 5790 7576 11 32 5790 7665 11 32 57917738 11 34 5792 7040 44 68 5793 7530 11 33 5794 7076 47 70 5795 7988 323 5796 6718 52 75 5797 7336 184 204 5798 7073 43 64 5798 8001 33 545799 7499 11 32 5800 8385 218 238 5801 6865 117 137 5801 7524 117 1375802 7037 96 117 5803 7822 36 56 5804 8145 114 137 5805 7398 36 59 58067992 11 34 5807 6820 40 63 5808 7205 82 103 5809 7899 11 34 5810 7490 5881 5811 6833 11 32 5811 7727 11 32 5812 7408 11 31 5813 6848 11 31 58146941 192 211 5815 7069 37 58 5815 8368 37 58 5816 8357 11 31 5817 810811 30 5818 7814 11 30 5819 7529 28 48 5819 8248 28 48 5820 6763 47 665821 8012 11 30 5821 8091 11 30 5822 8054 54 73 5823 7268 239 259 58246732 11 34 5824 6827 11 34 5824 7184 11 34 5825 8340 11 30 5826 7300 1134 5827 8050 11 30 5828 8245 68 90 5829 7981 11 33 5829 7983 11 33 58307331 185 205 5831 7862 35 54 5832 7642 57 80 5832 8266 57 80 5833 824740 61 5834 7931 11 30 5834 8359 11 30 5835 7697 35 56 5836 7759 11 335837 8187 41 64 5838 7279 44 63 5839 8038 189 210 5839 8200 189 210 58407860 2 22 5841 6959 11 34 5841 7462 11 34 5842 6784 52 71 5842 8378 1130 5843 6772 73 96 5843 7181 73 96 5844 7932 155 178 5845 8074 11 325846 8306 11 30 5847 7202 38 61 5848 6780 41 61 5849 7833 11 34 58507619 11 31 5851 6819 11 34 5851 7274 38 61 5851 8144 38 61 5851 8386 3861 5852 6787 34 55 5852 8189 34 55 5853 7247 11 31 5854 6938 143 1645855 7191 168 188 5855 7316 168 188 5856 7737 76 97 5857 7918 44 67 58587551 11 34 5859 7774 11 34 5860 7375 11 33 5861 7745 132 152 5862 8211181 203 5863 7652 132 155 5864 7355 11 33 5865 7927 42 61 5866 7434 196218 5867 8039 67 86 5868 6731 11 34 5869 6929 63 84 5870 7864 7 27 58718133 30 50 5872 8292 211 230 5873 7226 11 34 5874 7237 66 89 5875 807911 33 5876 7508 172 191 5876 7913 159 178 5877 8263 137 161 5878 8083 1132 5879 8087 11 30 5880 7705 49 72 5881 7262 11 31 5881 7277 11 31 58818194 11 31 5882 7664 53 72 5883 6849 36 55 5883 7410 11 30 5884 7874 1132 5885 6948 11 32 5886 6756 11 31 5886 6837 11 31 5886 7016 11 31 58867654 11 31 5887 8159 48 68 5888 7948 79 98 5889 7353 32 52 5890 6972 5777 5891 8275 11 31 5892 7020 11 34 5893 7625 11 31 5894 7606 41 60 58947608 9 28 5895 6809 11 34 5896 6823 98 120 5896 6893 98 120 5896 7848100 122 5897 6795 11 30 5898 7843 11 32 5899 8298 11 32 5900 8363 11 345901 7248 10 33 5902 7885 11 31 5903 6875 11 35 5904 6975 112 133 59057461 11 34 5906 6951 46 69 5907 6877 11 35 5907 7390 11 35 5908 8199 7597 5909 7883 39 62 5910 7088 85 108 5911 6709 11 34 5912 7265 30 50 59127663 30 50 5912 8339 30 50 5913 6885 221 244 5914 8406 11 30 5915 755011 34 5916 7804 75 98 5917 8327 60 83 5918 7487 11 34 5919 7681 44 655920 7012 115 136 5921 7256 11 34 5922 7000 42 66 5923 8367 11 31 59246919 11 34 5925 7749 36 55 5926 8352 11 30 5927 7838 11 32 5928 7074 3857 5928 8063 38 57 5929 6826 40 60 5930 8109 125 144 5931 8387 76 965932 7042 11 31 5932 7049 11 31 5932 7210 11 31 5932 7875 11 31 59336764 11 31 5933 8365 11 31 5934 8202 115 136 5935 7991 11 30 5936 711811 31 5936 7850 11 31 5936 7851 11 31 5936 8141 11 31 5936 8213 11 315936 8228 11 31 5937 7115 11 32 5938 6713 11 30 5938 6947 11 30 59396976 134 155 5940 6984 11 34 5941 7857 11 32 5941 8075 11 32 5942 771338 57 5943 7622 50 69 5944 6852 11 31 5945 7528 11 33 5946 7954 54 745947 8198 11 34 5948 6910 236 257 5949 7975 11 31 5950 7844 230 251 59518094 73 93 5952 6892 11 31 5953 6703 130 151 5953 6789 130 151 5954 712711 33 5955 7728 73 96 5955 8208 73 96 5956 7801 52 74 5957 7612 11 315958 7213 33 52 5958 7768 33 52 5959 6786 40 60 5960 7633 11 32 59617790 11 31 5961 7950 11 31 5962 7351 11 34 5963 6995 54 77 5964 8027 4265 5964 8347 42 65 5965 7466 108 127 5966 7500 11 34 5967 7495 49 695968 7463 80 104 5968 8296 80 104 5969 7156 11 31 5969 7377 39 59 59708099 52 75 5971 6821 11 34 5971 7549 198 221 5971 7807 198 221 5971 7880198 221 5972 8253 11 35 5973 8154 107 130 5974 8214 11 31 5975 7986 1132 5976 7150 89 109 5977 7723 42 65 5978 6845 139 159 5978 6902 139 1595978 7275 11 31 5978 7707 140 160 5979 7887 11 30 5980 6710 86 107 59806998 86 107 5980 7302 86 107 5981 7855 11 33 5982 6722 46 67 5982 743646 67 5982 8023 46 67 5983 6930 202 224 5984 8364 42 63 5985 7392 48 675986 7100 42 61 5986 7208 42 61 5986 7209 42 61 5986 7569 42 61 59867865 42 61 5986 7866 42 61 5986 7867 42 61 5986 8053 42 61 5986 8244 4261 5987 7014 11 31 5988 7116 11 32 5988 8404 11 32 5989 6866 92 115 59907849 137 160 5991 7630 168 187 5991 8260 168 187 5992 6791 11 32 59938116 10 29 5994 7407 175 195 5995 7158 11 31 5996 8107 88 111 5997 7252188 209 5997 7675 182 203 5998 7106 38 58 5999 8045 46 69 6000 7224 5676 6001 7225 36 56 6001 7735 36 56 6002 6749 11 34 6003 7273 64 83 60046983 56 79 6004 7832 56 79 6005 6859 119 141 6006 7692 11 32 6007 777336 55 6008 8096 11 32 6009 8146 80 99 6010 7133 236 256 6010 7568 204224 6011 8078 11 34 6012 8092 241 261 6013 6816 211 232 6014 6817 11 356014 7909 11 35 6015 6705 35 55 6016 7379 45 68 6017 7762 68 92 60187027 11 35 6018 7173 11 35 6019 7819 11 32 6020 7192 11 35 6021 8119 1132 6022 6911 30 49 6023 7031 11 31 6024 7232 11 32 6025 6840 173 1946026 8062 160 180 6027 8035 54 78 6028 8186 11 33 6029 6880 39 59 60308076 33 53 6031 6992 11 30 6032 7230 119 141 6032 8229 11 33 6033 679857 76 6034 7064 201 222 6035 8383 65 86 6036 8289 11 34 6037 7082 11 316038 7648 48 70 6039 8407 200 220 6040 7095 54 77 6041 8163 11 34 60427501 45 69 6042 7580 45 69 6042 7962 45 69 6043 6955 11 31 6043 7446 1131 6043 7515 11 31 6043 7543 11 31 6043 7617 11 31 6043 7618 11 31 60437726 11 31 6043 8098 11 31 6043 8290 11 31 6044 7086 11 34 6044 7306 1134 6044 7736 11 34 6045 7620 231 252 6045 7906 231 252 6046 7321 11 336047 6967 11 31 6048 7101 11 30 6048 8333 11 30 6048 8362 11 30 60497216 11 30 6049 7706 11 30 6050 7481 11 33 6051 8040 11 33 6052 6807 1131 6053 7431 11 32 6053 8203 11 32 6054 7218 11 34 6054 7968 11 34 60558043 38 57 6056 8336 11 30 6057 8225 11 33 6058 6818 52 71 6059 7996 1134 6060 6918 11 32 6061 8090 11 32 6062 7021 11 30 6063 7441 51 71 60647667 385 408 6065 8273 50 72 6066 7046 11 34 6067 8021 31 50 6068 6769100 121 6068 7033 100 121 6069 7438 49 71 6070 6931 42 63 6070 7930 4263 6071 8384 11 30 6072 8349 11 30 6072 8350 11 30 6073 6944 11 30 60738315 11 30 6074 6862 11 30 6075 7748 11 32 6076 6974 73 93 6077 7393 1131 6077 7680 11 31 6077 7732 11 31 6077 7859 11 31 6078 6750 211 2306079 8376 11 30 6080 6792 11 33 6081 8281 11 32 6082 7552 11 30 60837168 47 70 6084 7443 45 68 6085 7061 11 31 6085 7583 35 55 6086 6738 4060 6087 7367 11 34 6088 8399 11 32 6089 6752 36 59 6090 6863 11 31 60917806 11 30 6092 7308 51 73 6092 7688 51 73 6092 8301 51 73 6093 7539 1133 6094 7380 58 79 6094 8293 58 79 6095 7135 11 31 6096 8257 11 34 60968291 11 34 6097 7129 34 53 6098 7747 11 31 6099 6943 86 106 6100 8182 1134 6100 8221 11 34 6101 6904 11 32 6101 8254 11 32 6102 7972 118 1426103 6693 11 32 6104 7190 82 101 6105 7673 11 34 6106 7142 11 34 61077892 11 31 6108 7672 34 53 6109 7628 11 31 6110 8282 11 33 6111 6935 1130 6111 7259 11 30 6111 7678 234 253 6112 6770 41 60 6112 7352 41 606113 7775 11 35 6114 7700 11 30 6115 6854 11 31 6115 7294 11 31 61168328 11 31 6117 7357 304 326 6118 7051 210 232 6119 7572 204 227 61208085 66 85 6121 7125 11 30 6121 7305 11 30 6122 8358 11 34 6123 7285 5373 6123 7312 53 73 6123 7458 52 72 6123 7502 53 73 6123 7544 53 73 61237545 53 73 6123 7643 53 73 6123 7717 53 73 6123 7718 53 73 6123 7719 5373 6123 7720 53 73 6123 7721 53 73 6123 7890 53 73 6123 8123 53 73 61238150 53 73 6123 8151 53 73 6123 8152 53 73 6123 8153 53 73 6124 7119 5376 6125 7141 11 33 6126 6895 11 31 6127 6923 11 30 6127 7188 11 30 61288112 51 74 6129 6867 36 58 6129 8343 36 58 6130 8127 69 89 6131 7915 1134 6132 6986 11 30 6133 7984 11 32 6134 7753 11 32 6135 6879 11 34 61368341 45 68 6137 7503 126 145 6138 6886 85 104 6139 6768 34 53 6139 691634 53 6140 7318 11 30 6141 7778 11 33 6142 8179 11 30 6143 6721 11 306143 8019 11 30 6144 7322 147 169 6145 7395 42 62 6146 7815 72 92 61478237 96 117 6148 7412 35 56 6149 6932 11 34 6150 6719 37 61 6150 6940 3761 6150 7361 37 61 6150 8267 38 62 6150 8286 37 61 6151 7605 11 35 61527378 37 60 6152 8366 11 34 6153 6771 11 31 6154 7112 11 32 6155 7035 89109 6155 7109 89 109 6156 7028 11 35 6156 7400 11 35 6156 8103 11 356157 7288 11 32 6157 8355 11 32 6158 8360 58 81 6159 8095 37 56 61607666 38 58 6161 7036 11 33 6162 8067 11 35 6163 7637 11 34 6164 6907 173195 6164 7401 173 195 6164 8031 173 195 6165 6900 11 31 6166 7989 11 346167 7249 11 31 6168 7038 157 176 6169 7270 49 68 6170 7547 6 27 61717985 11 31 6172 7782 11 34 6173 7769 11 34 6174 7595 11 34 6175 7953 1135 6176 8392 11 32 6177 7731 248 269 6177 8279 242 263 6178 7826 11 326179 6882 39 62 6179 7344 11 34 6179 7584 39 62 6179 7695 39 62 61798117 38 61 6180 7767 11 32 6180 7789 11 32 6180 7926 11 32 6181 7472 1130 6182 8389 70 89 6183 7370 11 31 6183 7878 11 31 6184 6794 71 93 61856697 11 31 6185 7337 11 31 6185 8294 11 31 6186 7881 43 62 6187 7777 1133 6188 6890 11 34 6189 7179 174 197 6190 6898 11 34 6191 7451 11 346192 7594 11 34 6193 7136 59 79 6193 8373 59 79 6193 8382 59 79 61948142 42 65 6195 6962 11 33 6195 6981 11 33 6195 7350 11 33 6195 7418 1133 6195 7536 11 33 6195 7537 11 33 6195 7538 11 33 6195 8391 11 33 61967114 100 121 6197 6776 104 123 6197 6810 104 123 6198 7198 11 32 61987222 11 32 6198 7924 51 72 6199 6689 72 93 6199 7342 72 93 6199 7498 7293 6200 7236 11 31 6201 8052 11 34 6202 7235 11 35 6202 8162 11 35 62028169 11 35 6203 7320 11 34 6204 6980 11 31 6205 7123 11 30 6206 7559 304325 6207 6950 43 62 6207 6987 43 62 6207 7858 11 30 6208 7493 11 33 62098240 203 222 6210 7011 11 34 6211 7982 37 57 6212 6758 11 30 6212 830411 30 6213 6777 45 68 6214 7052 40 59 6215 7137 46 68 6215 7139 47 696216 7382 11 30 6217 6997 33 52 6218 7267 11 31 6218 7387 51 71 62187419 51 71 6218 7518 11 31 6218 7527 51 71 6218 7795 11 31 6218 7908 5171 6218 7998 51 71 6218 8114 11 31 6218 8184 51 71 6218 8335 11 31 62198201 11 34 6220 7621 11 34 6221 7098 11 31 6222 7847 47 68 6223 7896 3353 6224 8051 11 34 6225 8231 49 72 6226 8036 10 33 6227 7448 41 64 62287990 1 24 6229 7684 137 160 6230 6737 11 34 6230 7155 11 34 6231 7473 1134 6232 7024 44 63 6232 8010 43 62 6232 8329 44 63 6233 6908 41 60 62348274 40 63 6235 8136 11 30 6236 6726 137 160 6237 8110 11 34 6238 788438 59 6239 7995 11 30 6240 8000 46 69 6241 7751 11 34 6242 6702 11 306242 8331 11 30 6242 8397 11 30 6243 7054 75 95 6243 7346 75 95 62447050 11 34 6245 6855 71 90 6245 8370 71 90 6246 7900 84 106 6247 6846112 135 6248 7579 47 68 6248 7734 47 68 6249 7278 128 148 6250 7146 1130 6251 7708 38 58 6252 7313 47 70 6253 6699 11 34 6254 7199 72 93 62556868 49 69 6255 7362 49 69 6255 7604 49 69 6256 7977 39 62 6257 8302 1131 6258 8196 11 31 6259 7108 11 34 6260 8243 236 259 6261 7284 11 326261 7746 11 32 6262 7349 38 58 6262 7402 11 31 6263 7261 33 55 62637629 34 56 6263 8309 33 55 6264 7189 11 34 6265 6899 11 34 6266 6889 3352 6267 6937 11 35 6268 7455 11 31 6269 6796 11 31 6269 7656 11 31 62706897 78 102 6271 7102 35 55 6271 8197 35 55 6272 6825 86 109 6272 709086 109 6273 7961 53 75 6274 8044 54 74 6275 7077 34 55 6276 7203 95 1166277 7478 71 91 6278 7494 177 200 6278 7999 11 34 6279 7870 11 34 62807877 11 34 6281 7505 11 34 6281 7533 11 34 6281 7534 11 34 6282 7325 82103 6283 7694 190 209 6284 6994 31 51 6284 8061 31 51 6285 6887 48 706286 7456 141 164 6287 7489 46 67 6288 7846 51 72 6289 7659 11 34 62907405 47 70 6290 7955 47 70 6290 7960 47 70 6291 7651 36 55 6291 7904 6180 6292 8168 52 73 6293 7743 11 30 6294 7519 11 32 6295 7238 11 30 62957385 11 30 6295 7903 11 30 6296 7159 11 30 6297 7214 11 31 6298 8297 4262 6299 7886 44 63 6300 6936 170 189 6301 8118 134 156 6302 8177 376 3976303 7776 11 34 6304 7081 40 61 6305 7348 42 65 6306 7586 59 82 63076963 112 132 6308 6805 11 35 6308 7257 11 35 6309 7332 83 102 6310 7825237 260 6311 7333 11 34 6311 7334 11 34 6312 6802 11 35 6312 6925 11 356312 7080 11 35 6312 7592 11 35 6312 7596 11 35 6312 7600 11 35 63127639 11 35 6312 7699 11 35 6312 7702 11 35 6312 7733 11 35 6312 7842 1135 6312 7923 11 35 6312 8081 11 35 6312 8093 11 35 6312 8183 11 35 63136808 11 31 6313 6828 11 31 6314 7065 11 32 6314 7066 11 32 6314 7067 1132 6314 8388 11 32 6315 7425 213 234 6316 7509 11 34 6317 7416 11 306318 7788 92 111 6319 6891 34 54 6319 7169 151 171 6320 6753 79 99 63217250 31 51 6322 6806 52 75 6323 8319 305 325 6324 7365 55 75 6325 713111 34 6326 7764 38 57 6327 6922 11 30 6328 8018 141 160 6329 7742 98 1176330 7187 11 31 6331 6969 70 93 6332 7391 62 85 6333 8402 236 258 63347898 37 60 6335 7426 11 32 6336 7366 217 236 6337 6888 11 34 6338 7486189 211 6339 6953 11 32 6339 7925 11 32 6340 8165 11 32 6341 6839 11 336342 7079 242 262 6343 7289 11 31 6343 7852 11 31 6344 7750 11 31 63457657 10 30 6345 8251 11 31 6346 7107 11 33 6347 7180 240 262 6347 8219236 258 6348 7800 11 30 6349 8288 218 237 6350 6954 11 30 6351 6985 4366 6352 7624 48 68 6353 7269 11 31 6354 7007 11 33 6355 6961 208 2286355 8265 208 228 6356 6691 11 34 6357 7315 11 31 6358 6751 196 215 63596759 11 31 6360 8206 200 222 6360 8351 200 222 6361 7609 11 31 6362 749211 32 6363 8371 350 374 6364 6927 116 135 6365 7104 230 251 6366 7255 1131 6367 7126 64 83 6368 7980 11 33 6369 7602 11 32 6370 7854 11 33 63718157 11 31 6372 7570 136 156 6373 7343 52 71 6374 8002 11 30 6374 802211 30 6375 6746 43 63 6375 7004 43 63 6375 7157 11 31 6375 7384 43 636375 7939 43 63 6375 8374 11 31 6376 7987 11 32 6377 7471 11 33 63777480 52 74 6377 7689 51 73 6378 7111 11 34 6379 7097 167 187 6379 766211 31 6380 7701 11 30 6381 7974 95 118 6382 7062 62 85 6383 8220 69 916384 7347 40 60 6384 8223 40 60 6385 7113 40 64 6385 7164 40 64 63857729 11 35 6386 6982 58 78 6387 7083 65 89 6387 7946 70 94 6387 8316 7195 6388 7645 11 34 6389 6896 11 35 6390 7071 35 55 6390 7084 35 55 63916801 48 72 6392 7722 11 30 6393 7582 219 242 6394 7172 240 262 6395 840511 34 6396 7949 65 87 6397 8401 82 103 6398 8097 37 59 6399 6701 47 706400 7075 11 32 6401 7298 11 34 6402 8059 35 56 6403 7301 11 30 64038256 11 30 6404 7120 30 50 6405 7258 11 33 6406 7263 11 31 6407 8003 1134 6408 6876 11 31 6409 6914 11 31 6409 7938 11 31 6410 6779 32 52 64107364 32 52 6411 7043 11 32 6411 7797 11 32 6412 6883 11 30 6413 6733 4668 6414 7467 81 101 6415 7422 11 31 6415 7766 11 31 6416 7148 34 53 64177661 11 33 6418 6757 11 31 6418 7468 11 31 6419 6834 37 56 6420 6851 1135 6420 6971 11 35 6420 7311 11 35 6420 7374 11 35 6420 7626 11 35 64207784 11 35 6420 7799 11 35 6420 8313 11 35 6420 8338 11 35 6421 7017 215238 6422 7053 125 148 6422 7185 11 34 6423 8325 11 30 6424 7757 11 306425 7211 32 52 6426 7976 389 412 6427 8037 49 73 6428 7916 11 30 64298342 33 54 6430 7531 11 31 6431 7830 3 25 6432 7411 133 155 6433 6965 4564 6433 7449 46 65 6433 7562 45 64 6433 8033 11 30 6434 7791 155 1766435 7516 134 157 6436 7389 69 89 6437 8334 76 96 6438 7147 11 30 64387147 54 73 6438 8269 11 30 6439 7272 11 30 6439 8271 11 30 6440 8227 5275 6441 7636 11 34 6442 7593 11 34 6442 7593 67 90 6442 8381 11 34 64428381 67 90 6443 7818 46 69 6443 8077 44 67 6444 7676 11 34 6444 7808 1134 6445 7420 11 31 6446 6814 138 157 6447 6728 202 222 6447 7368 201 2216448 7535 11 34 6449 6822 1 24 6449 7513 11 34 6450 8249 47 70 6451 705611 34 6452 7309 98 119 6453 7299 32 51 6453 7623 26 45 6454 8104 71 936455 7291 51 72 6455 7292 51 72 6456 7658 31 50 6457 7099 11 34 64578132 11 34 6457 8139 11 34 6458 8258 11 30 6459 6841 60 84 6460 6901 5172 6461 8082 11 30 6462 7640 11 34 6463 7091 11 31 6464 8070 11 32 64657329 62 81 6465 7574 62 81 6465 7716 62 81 6465 7935 62 81 6465 8317 6281 6465 8353 62 81 6465 8369 62 81 6466 7597 70 90 6467 7437 221 2446467 7541 75 98 6468 6874 11 30 6468 6945 11 30 6468 7153 11 30 64697803 192 213 6470 7940 11 34 6470 7940 67 90 6471 6727 11 32 6471 691311 32 6472 7432 2 23 6473 7345 11 31 6474 7703 62 85 6475 7969 30 496476 7130 35 57 6477 7162 251 272 6478 7201 74 98 6478 7413 11 35 64787421 74 98 6478 7452 74 98 6478 7567 74 98 6478 7963 74 98 6479 7952 1130 6480 7128 68 92 6481 7610 11 34 6482 8121 11 32 6483 7506 11 32 64837787 11 32 6483 7901 11 32 6484 8318 42 63 6484 8346 42 63 6485 7372 1134 6486 7369 11 35 6487 7805 87 106 6488 6915 36 59 6489 7444 60 82 64906857 11 34 6490 7376 11 34 6490 8181 11 34 6491 6783 11 32 6491 7429 1132 6491 7889 10 31 6491 8167 11 32 6492 7589 196 215 6493 7911 62 836494 7231 11 31 6495 7371 11 34 6496 6956 11 34 6496 8007 11 34 64978207 11 33 6498 7006 11 34 6499 7341 189 209 6500 7682 45 69 6501 767411 35 6501 7712 11 35 6502 7008 11 33 6503 7465 170 191 6504 7022 11 306505 6920 47 66 6505 8311 47 66 6506 7304 60 79 6507 6785 92 111 65078105 29 48 6507 8134 28 47 6508 7542 71 94 6509 6714 41 64 6509 7894 4164 6509 7959 42 65 6510 7761 186 209 6511 7603 250 273 6512 7254 11 306513 7497 240 262 6514 7445 41 60 6515 7200 169 189 6515 7280 169 1896515 7649 169 189 6516 6973 11 32 6516 7327 11 32 6516 7957 11 32 65178014 41 63 6518 7942 81 104 6519 7186 11 30 6520 7464 61 84 6521 7687 1132 6522 6988 11 30 6523 6843 11 30 6524 7013 11 32 6525 6844 11 32 65258188 11 32 6525 8320 11 32 6526 7328 143 164 6527 6799 11 32 6528 727111 32 6529 7242 11 32 6530 8138 11 34 6531 6742 11 30 6532 7399 11 316533 7607 176 195 6534 7171 3 22 6535 7029 41 62 6535 7176 41 62 65357195 41 62 6535 7196 11 32 6535 8101 108 129 6536 7424 11 31 6537 681111 34 6538 7917 11 32 6539 7039 11 32 6539 7244 11 32 6539 7647 11 326540 8324 174 197 6541 6933 11 34 6542 6704 11 33 6542 6773 11 33 65427160 11 33 6542 7970 11 33 6543 6946 11 31 6544 7229 11 30 6544 8361 1130 6545 7151 11 32 6546 7260 3 24 6547 7561 11 32 6548 8190 11 31 65488218 11 31 6549 8072 11 34 6550 7563 34 57 6551 8226 11 31 6552 6747 1134 6553 7827 11 30 6554 6871 86 107 6554 7783 86 107 6555 7598 11 346556 7941 9 29 6556 8060 9 29 6557 7479 8 30 6558 7861 11 34 6559 7876120 143 6560 7282 90 112 6560 7283 185 207 6561 6979 11 34 6561 7219 1134 6562 7215 35 58 6563 6695 11 34 6563 6743 57 80 6563 6835 11 34 65637070 57 80 6563 7453 11 34 6563 7724 11 34 6563 7740 11 34 6563 7741 1134 6563 7824 11 34 6563 8086 57 80 6563 8140 57 80 6563 8321 11 34 65646706 11 30 6565 6755 101 122 6565 6797 100 121 6565 7725 101 122 65658191 101 122 6566 7002 11 35 6567 8280 11 34 6568 7212 61 80 6569 702311 31 6569 7693 11 31 6569 7994 11 31 6569 8160 11 31 6569 8161 11 316570 8323 11 32 6571 8322 84 107 6572 7307 11 34 6573 7796 11 34 65747683 11 34 6575 6803 195 215 6576 7967 11 33 6577 7557 55 78 6578 7403 223 6579 6996 11 34 6579 7415 46 69 6579 7714 11 34 6580 8238 11 35 65816700 11 33 6582 6696 35 55 6583 7103 11 34 6584 7161 34 54 6585 8242 1133 6586 7433 100 123 6587 7386 11 30 6588 8332 95 118 6589 8224 11 316590 7019 11 32 6590 8277 11 32 6591 7094 11 31 6592 7785 11 33 65937792 211 234 6594 7951 11 30 6595 7560 39 59 6596 6903 219 240 6597 693911 30 6598 6800 11 34 6599 8379 41 65 6600 6999 11 30 6600 7690 11 306601 6717 11 34 6601 7679 11 34 6602 7409 224 244 6603 8212 34 53 66047615 11 32 6604 7616 11 32 6604 7671 11 32 6604 7696 11 32 6605 6831 91115 6605 8088 91 115 6606 8071 37 60 6607 6964 11 31 6608 8398 143 1626609 8089 45 65 6610 8020 11 34 6611 8299 11 31 6612 6692 130 153 66126872 130 153 6613 6928 41 64 6614 7145 11 35 6615 7758 71 90 6616 7711172 195 6617 8073 79 102 6618 6711 104 125 6619 6782 42 61 6620 7251 4161 6621 7770 11 30 6622 7943 61 85 6623 7837 33 52 6624 6761 11 31 66258262 11 32 6626 7122 52 75 6627 6729 11 32 6627 7178 11 32 6628 7548 3858 6629 7739 11 32 6629 8252 11 32 6630 8261 11 32 6630 8295 204 2256631 7578 57 78 6632 7047 41 60 6632 7234 41 60 6632 7522 41 60 66337221 11 31 6634 7482 11 32 6634 8068 11 32 6635 8005 11 33 6636 8137 5073 6637 6842 11 31 6638 6813 31 51 6638 7286 31 51 6638 8006 31 51 66396745 11 31 6639 7241 11 31 6639 7454 4 24 6640 7338 11 34 6641 7811 166186 6642 8283 82 103 6643 6812 11 34 6644 8102 11 31 6645 6775 11 316646 7044 11 30 6647 6838 165 185 6648 6684 54 74 6649 7048 196 219 66507794 78 99 6651 8216 86 106 6652 8009 226 246 6653 6740 11 31 6653 714911 31 6653 8126 11 31 6653 8395 11 31 6654 7281 7 30 6655 7096 11 316655 7993 11 31 6656 7339 187 210 6657 6788 11 31 6657 6926 106 126 66577856 45 65 6657 8055 94 114 6658 8380 43 66 6659 6735 207 227 6659 7276125 145 6659 7553 11 31 6660 6712 34 57 6661 7523 11 35 6662 8106 44 676663 6688 115 136 6664 7929 6 29 6665 6744 11 34 6666 8232 208 228 66678028 48 70 6668 7032 174 195 6669 8400 11 34 6670 8069 39 62 6671 754011 32 6672 7058 11 34 6673 8015 73 96 6674 6917 11 34 6675 6715 61 816675 6960 61 81 6675 7228 61 81 6676 6966 11 34 6677 7863 84 107 66787404 98 121 6678 7973 11 34 6678 8173 11 34 6679 7174 11 32 6680 8300 1131 6681 7831 41 64 6682 8234 240 263 6683 8113 11 32 6683 8372 11 328409 8604 11 31 8409 8626 11 31 8410 8649 11 31 8411 8660 104 123 84128674 11 30 8413 8655 11 31 8414 8671 129 149 8415 8594 70 90 8415 874070 90 8416 8713 11 31 8417 8648 129 149 8418 8562 11 30 8418 8588 11 308418 8592 70 89 8419 8579 11 31 8420 8571 70 90 8420 8634 75 95 84218584 11 31 8422 8572 99 118 8422 8590 76 95 8423 8612 11 30 8424 8619 3050 8425 8717 11 32 8426 8639 11 31 8427 8573 146 166 8428 8684 376 3968429 8650 158 177 8430 8568 11 32 8430 8576 11 32 8430 8664 11 32 84318600 11 33 8432 8718 699 719 8433 8723 11 31 8434 8561 11 33 8435 872962 84 8436 8635 11 34 8437 8678 36 57 8438 8694 45 68 8439 8589 11 318440 8631 80 100 8441 8685 11 31 8442 8616 67 87 8443 8574 11 31 84448581 11 32 8445 8712 57 78 8446 8659 61 81 8447 8567 11 31 8448 8632 7596 8449 8643 53 73 8450 8677 176 196 8451 8690 11 31 8452 8687 11 338453 8585 11 34 8454 8599 11 31 8455 8665 11 30 8456 8647 208 228 84578668 184 204 8458 8722 64 84 8459 8666 11 32 8460 8610 11 33 8461 8570217 240 8462 8636 11 30 8463 8692 164 184 8464 8719 242 261 8465 8691 5878 8466 8607 11 30 8466 8622 11 30 8467 8680 11 31 8468 8653 11 31 84698708 167 187 8470 8706 11 35 8471 8583 115 135 8472 8564 99 118 84728566 101 120 8472 8577 101 120 8472 8645 94 113 8472 8658 101 120 84728661 94 113 8473 8595 11 35 8474 8725 11 31 8475 8670 96 119 8476 859611 30 8477 8615 638 658 8478 8704 11 34 8479 8625 57 81 8480 8731 84 1048481 8608 11 30 8482 8733 11 34 8483 8672 227 250 8484 8624 11 33 84858703 117 140 8486 8667 11 30 8487 8621 40 59 8488 8580 11 34 8489 861811 32 8490 8644 53 74 8491 8720 11 31 8492 8628 46 69 8493 8686 11 328494 8697 49 68 8495 8732 55 74 8496 8614 122 144 8497 8591 53 73 84988652 11 32 8499 8693 60 80 8500 8651 8 27 8501 8663 11 31 8502 8586 1131 8503 8739 11 30 8504 8605 187 210 8504 8737 185 208 8505 8657 77 998506 8711 11 31 8507 8702 11 30 8508 8727 11 31 8509 8617 130 149 85098640 231 250 8510 8709 149 168 8511 8565 60 80 8512 8593 79 99 8512 872179 99 8512 8728 79 99 8513 8679 152 172 8514 8642 11 30 8515 8601 118138 8516 8736 63 82 8517 8633 11 31 8518 8701 11 30 8519 8700 42 62 85208623 11 30 8521 8630 11 30 8522 8682 176 199 8523 8646 11 32 8524 860211 31 8525 8724 11 30 8526 8716 115 134 8527 8669 39 60 8528 8688 11 308529 8627 11 31 8530 8689 93 117 8530 8734 92 116 8531 8673 11 31 85328705 151 172 8533 8698 11 31 8534 8629 11 32 8535 8613 376 395 8536 860911 31 8537 8741 67 86 8538 8578 11 31 8538 8654 11 31 8539 8597 126 1468540 8598 155 174 8541 8582 11 32 8542 8587 61 81 8543 8707 166 186 85448637 64 84 8544 8638 64 84 8544 8699 64 84 8545 8641 73 93 8546 8726 6485 8547 8715 11 31 8548 8575 11 31 8549 8620 11 32 8550 8603 11 31 85518735 158 178 8552 8681 75 95 8553 8676 11 31 8553 8730 11 31 8554 868311 31 8555 8675 100 120 8556 8611 76 96 8556 8662 76 96 8556 8695 76 968556 8710 76 96 8557 8656 355 375 8558 8696 11 32 8558 8714 11 32 85598563 11 30 8559 8606 11 30 8559 8738 11 30 8560 8569 11 32

MicroRNAs and their precursors and promoters, especially those having adifferential expression pattern between water-sufficient andwater-insufficient (drought or water stress) conditions, are useful inengineering desirable traits (e.g., increased yield, improvedgermination) in crops that can experience water stress. Similar utilityis found in other miRNAs (and their precursors or promoters) havingexpression patterns specific to other abiotic or biotic stressconditions, e.g., miRNAs having a differential expression patternbetween nutrient-sufficient and nutrient-insufficient conditions, orbetween thermally stressed and thermally non-stressed conditions.Suitable methods include the introduction of an exogenous miRNArecognition site into a sequence, deletion or modification of anendogenous miRNA recognition site from a sequence, engineering of anative miRNA or miRNA precursor sequence in order to recognize asequence other than the endogenous target sequence, and use of a miRNApromoter to provide a particular expression pattern.

Example 6

This example describes identification of a crop plant miRNA (miRMON18)having a specific expression pattern characterized by strong expressionunder nitrogen-sufficient conditions and suppression undernitrogen-deficient conditions, or strong expression underphosphate-sufficient conditions and suppression underphosphate-deficient conditions

Small RNAs were cloned and putative miRNAs identified from a variety oftissues and developmental stages from rice (Oryza sativa cv.Nipponbare), corn (maize, Zea mays var. LH244), and soybean (Glycine maxvar. A3525), following techniques described above in Examples 1 and 5.Small RNA abundances were normalized between libraries and calculated astranscripts per quarter million sequences (tpq). A putative mature miRNA(small RNA number 370903, assigned the trivial name “miRMON18”) with thesequence UUAGAUGACCAUCAGCAAACA was identified in rice (SEQ ID NO. 393),maize (SEQ ID NO. 3227), and soybean (SEQ ID NO. 8742) small RNAlibraries. This sequence did not match known miRNAs in miRBase.

A miRMON18 precursor sequence was identified from the rice genome as

(SEQ ID NO. 1763, FIG. 7B)CCAUGAACCUGUUUUGUUGCUGGUCAUCUAGCUACCCGUGCAUGCCUGGAGAUUGGAGAAUAAUUGACGAUGCAGCAGUCGGCUUAUUGGCUCUUGGGCACGCGUGGUUAGAUGACCAUCAGCAAACAAGUUCGUGAG,.Another putative miRMON18 precursor sequence was identified fromavailable maize genomic data as

(SEQ ID NO. 3936, FIG. 7A)CUCCGAACCUGUUUUGUUGGUGGUCAUUUAACCAUGCAUGCUUCGAUCGAUGGAUUGGUGCAUGCAUGGAUUAUUGCAUAGUGUGAUGCAUGUGGCGCAUCAGUGCAUGGUUAGAUGACCAUCAGCAAACAUGUUCUUGAG.The position of the mature miRMON18 is depicted above in underlined textin these precursor sequences (SEQ ID NO. 1763 and SEQ ID NO. 3936). EachmiRMON18 precursor was predicted to form a fold-back structure (FIGS. 7Aand 7B), with the mature miRNA (miRMON18) located in the 3′ arm of thefold-back structure, with the predicted miRNA* (“miRMON18*”) located inthe 5′ arm; this was consistent with the much greater abundance of themature miRMON18 relative to that of miRMON18* observed in the singlecorn locus MRT4577_(—)378723C.3. A fold-back structure having thesequence

(SEQ ID NO. 8743, FIG. 7C)UGCAACCCUUGAAUGUGUUUGUUGAUUGAUAUCUACACAUGUUGAUCAUCCUUGUGUUGAUCGAUUGGUUUAGAUGACCAUCAACAAACUCUUUCGUGGU UUUGCAwas identified in Arabidopsis thaliana as the precursor to a relatedmature miRNA with the sequence UUAGAUGACCAUCAACAAACU (miR827, SEQ ID NO.8744). The mature miR827 was observed only at low abundance inArabidopsis thaliana. Alignment of the two mature miRNAs shows thatmiR827 differs from miRMON18 by two nucleotides (FIG. 7D). This twonucleotide difference between miRMON18 and miR827 appears to beconserved between monocots and dicots, with miRMON18 identified in maize(Zea mays), rice (Oryza sativa), and sugar cane (Saccharum officinarum,data not shown) and miR827 identified in dicots (Arabidopsis thaliana).

Northern blots verified expression of the miRMON18 21-mer in at leastrice (grain and seedling) and maize (kernel, leaf, and root) tissuesamples from plants grown under normal (non-stressed) conditions, asdepicted in FIG. 8A. MicroRNA precursors originate as polyadenylatedtranscripts generated by RNA polymerase II and standard transcriptionprofiling of the maize miRMON18 precursor (SEQ ID NO. 3936,corresponding to probeset A1ZMO68928_at) further confirmed elevatedexpression in maize endosperm, callus, and seedling (FIG. 8B), withexpression in other tissues in this sample falling below the detectioncut-off threshold of 500 units.

Expression of the maize miRMON18 precursor (SEQ ID NO. 3936) wasanalyzed in maize tissues from plants grown under water-deficient(drought) (FIG. 9A), cold (FIG. 9B), and nitrogen-deficient conditions(FIG. 9C). Expression of the miRMON18 precursor was relativelyunaffected by water conditions in root, shoot, ear, kernel, and tassel,with expression in leaf and silk increased under water-deficientconditions relative to water-sufficient conditions (FIG. 9A); expressionwas also relatively unaffected by temperature (FIG. 9B). However,miRMON18 expression was highly responsive to nitrogen conditions, withabout 2-fold suppression observed with nitrogen limitation (2 millimolarammonium nitrate), relative to expression observed with sufficientnitrogen (20 millimolar ammonium nitrate) (FIG. 9C). Thisnitrogen-responsive expression pattern was verified by phosphor imagequantification of Northern blots of small RNA samples, which showed anaverage 9.6-fold suppression of the mature miRMON18 21-mer over 3 timepoints (FIG. 10A). Thus, miRMON18 showed overall enhanced expression inmaize endosperm and kernel, and strong suppression in leaves induced bynitrogen deficiency.

In another experiment, maize was grown in a hydroponic system undersufficient phosphate until the V3 stage, then phosphate deprived for upto 3 days. Leaf tissue samples were taken at 1 and 3 days afterphosphate deprivation had begun. At 3 days, plants were returned tophosphate sufficiency and samples taken at 30 minutes and 6 hours afterrecovery. Control samples at each time point were taken from plantsgrown continually under phosphate sufficiency. FIG. 10B depicts theresults of northern blots analyzed with a miRMON18 probe anddemonstrates that, in this experiment, the maize miRMON18 mature miRNAexhibited in leaf tissue strong expression under phosphate-sufficientconditions and suppression under phosphate-deficient conditions.

Example 7

This example describes identification of genes having miRNA recognitionsites (miRMON18 recognition sites) natively regulated by a crop plantmiRNA (miRMON18) having an expression pattern characterized by strongexpression under nitrogen-sufficient conditions and suppression undernitrogen-deficient conditions, or strong expression underphosphate-sufficient conditions and suppression underphosphate-deficient conditions.

Putative targets for the mature miRMON18 (UUAGAUGACCAUCAGCAAACA, SEQ IDNO. 393, SEQ ID NO. 3227, or SEQ ID NO. 8742) were identified andincluded a clade of genes in the SPX (“SYG1/Pho81/XPR1”) domain family.The SPX domain has been assigned the protein family/domain identifierPfam PF03105, and is a hydrophobic domain found in the N-terminus ofseveral proteins, typically including a stretch of about 180 residueswith three smaller sub-domains of 35-47 amino acids; see, e.g., the SPXentry for the Pfam database currently curated at the Janelia FarmsResearch Campus of the Howard Hughes Medical Institute, publiclyavailable at pfam.janelia.org/family?acc=PF03105.

The majority of proteins in the SPX domain family include otherconserved domains in their C-terminus. For example, several proteins inthe SPX domain family also include in their C-terminus an EXS (“ERD1,XPR1, and SYG1”) domain, Pfam PF03124, which is possibly involved inprotein sorting; see, e.g., pfam.janelia.org/family?acc=PF03124. OtherSPX proteins include a conserved VTC (vacuolar transporter chaperone 2)domain, Pfam PF09359; see, e.g., pfam.janelia.org/family?acc=PF09359.Several SPX proteins include a conserved MFS_(—)1 or MFS (“majorfacilitator superfamily”) domain, Pfam PF07690, which is involved intransporting small solutes such as small sugars and inorganic salts inresponse to chemiosmotic ion gradients; seepfam.janelia.org/family?acc=PF07690. The SPX domain is likely to be atranscription factor, and may function as a dimerization domain.

SPX proteins include those encoded by the PHO genes, which are involvedin the loading of inorganic phosphate into the xylem of roots; see, e,g., Wang et al. (2004) Plant Physiol., 135:400-411, who have describedidentification of several PHO1 homologues, conservation of the SPXdomain within these proteins, and the PHO1 promoter's predominantexpression in the vascular tissues of roots, leaves, stems, or flowersas well as in some nonvascular tissues. Proteins in the SPX domainfamily are possibly involved in G-protein associated signal transduction(and thus are possibly sensors of inorganic phosphate); see Ticconi andAbel (2004) Trends Plant Sci., 9:548-555. The PHO1 genes include boththe SPX domain and an EXS domain. Members of the PHO clade also includea RING domain (At1g02860 and At2g38920), or an MFS domain (At4g22990,At4g11810, and At1g63010); see Wang et al. (2004) Plant Physiol.,135:400-411, especially FIG. 3 and FIG. 6 d.

Recently, a gene named NLA was reported to be required for adaptation tolow nitrogen availability in Arabidopsis thaliana; see Peng et al.(2007) Plant Cell, 50:320-337. NLA (“AtNLA”, locus At1g02860), assignedUniProtKB/Swiss-Prot accession number Q2V4F9, has the sequence ofMRT3702_(—)101115C, SEQ ID NO. 8745; annotation of the NLA protein ispublicly available at beta.uniprot.org/uniprot/Q2V4F9 and atpfam.janelia.org/protein?id=Q94C80_ARATH. The Arabidopsis NLA genehaving the sequence of SEQ ID NO. 8745 contains an SPX domain, an MFS 1domain, and a RING domain, and includes a miR827 recognition site(target) sequence TGTTTGTTGATGGTCATCTAA (SEQ ID NO. 8746) located atnucleotide positions 135 through 155, which was validated as a targetfor the Arabidopsis miR827. The NLA encodes a C3HC4-type RING-fingerubiquitin ligase (AT1G02860.1, SEQ ID NO. 8747); mutating this genedisrupts the adaptability of Arabidopsis to nitrogen limitation.

Ten additional clones of the AtNLA gene were sequenced. Clones 1, 4, and5 contained a partial AtNLA sequence (SEQ ID NO. 8748). Clone 2contained an AtNLA sequence lacking the SPX domain (SEQ ID NO. 8749).Clone 3 contained an AtNLA sequence lacking the RING domain (SEQ ID NO.8750). Clone 6 contained a genomic AtNLA fragment (SEQ ID NO. 8751) witha disrupted miR827 recognition site (target sequence) located atnucleotide positions 2142-2162. Clone 7 contained another AtNLA sequence(At1g63010, SEQ ID NO. 8752). Clone 8 contained another genomic AtNLAfragment (At1g63010, SEQ ID NO. 8753) with a disrupted miR827recognition site (target sequence) located at nucleotide positions2142-2162. Clone 9 contained another AtNLA sequence (At1g63010, SEQ IDNO. 8754) lacking the SPX domain. Clone 10 contained an AtNLA sequence(At1g63010, SEQ ID NO. 8755) lacking the MFS domain.

A number of “virtual” cDNAs were assembled from maize genomic and cDNAsequences, describing independent genes targeted by miRMON18. The firstof these novel miRMON18 targets (“SPX_MFS_(—)117961287”, derived fromBAC at GI:117961287) had the sequence of SEQ ID NO. 8756 and included anATG start codon at nucleotide positions 326-328 and a TGA stop codon atnucleotide positions 2414-2416; the longest open reading frame(translation frame=2) encoded by SEQ ID NO. 8756 had the amino acidsequence of SEQ ID NO. 8757. An alternatively spliced version of thisfirst novel miRMON18 target gene is SEQ ID NO. 8758(“SPX_MFS_(—)117961287_(—)2”), which includes an ATG start codon atnucleotide positions 87-89 and a TGA stop codon at nucleotide positions1137-1139; the longest open reading frame (translation frame=3) encodedby SEQ ID NO. 8758 had the amino acid sequence of SEQ ID NO. 8759.

The second of these novel miRMON18 targets had the sequence of SEQ IDNO. 8760 (“SPX_MFS2”, derived from BAC at GI:118200525) and included anATG start codon at nucleotide positions 201-203 and a TGA stop codon atnucleotide positions 2295-2297; the longest open reading frame(translation frame=3) encoded by SEQ ID NO. 8760 had the amino acidsequence of SEQ ID NO. 8761. An alternatively spliced version of thissecond novel miRMON18 target gene is SEQ ID NO. 8762(“SPX_MFS_(—)117961287_(—)2”), which includes an ATG start codon atnucleotide positions 145-147 and a TGA stop codon at nucleotidepositions 1189-1191; the longest open reading frame (translationframe=1) encoded by SEQ ID NO. 8762 had the amino acid sequence of SEQID NO. 8763.

A third novel miRMON18 target included stitched cDNA sequences from ESTdata and had the sequence of SEQ ID NO. 8764 (derived from BAC at GI:126116193) and included two possible ATG start codons at nucleotidepositions 217-219 and 1034-1036 and a TGA stop codon at nucleotidepositions 2093-2095. Two proteins were predicted from the two possibleopen reading frames by homology; the first protein (predicted with aframe shift of 1) contained 625 amino acids and had the sequence of SEQID NO. 8765, and the second protein contained 353 amino acids and hadthe sequence of SEQ ID NO. 8766.

The peptides encoded by these novel maize miRMON18 target genes werealigned using ClustalW (version 1.82); the resulting multiple sequencealignment is depicted in FIG. 11, and shows the maize SPX domain(indicated by underlined sequence, where present) and the maize MFSdomain (indicated by sequence in bold text).

Additional cloning work from BAC115312385 confirmed the sequence of thefirst miRMON18 target (“SPX_MFS_(—)117961287”, SEQ ID NO. 8756) andyielded the genomic SPX_MFS2 sequence SEQ ID NO. 8767 in which wasfurther identified leader sequence (indicated by italicized text), 5′introns (indicated by underlined text), exons (indicated by upper-casetext), and the miRMON18 recognition site (located at nucleotides2628-2648 of SEQ ID NO. 8767 and indicated by bold upper-case text):

(SEQ ID NO. 8767)gttacaaggcaatatttttgtagaataaaatcttaaaggaaactcaactccacgaattggtcacttgcattaaatcatattgtgggtctcttttagttgcatcttaagatggcggcaacaagatttcaagcactttttatctagtgaccgcaatgcactggagataaataagaatccaaatattatttttgataaccttgacactatttaaatcttcttataagtgacgaagtagtttgatcaacaataaaaacgtatagatttcaacattttttgcgattgtaggatatatgttagcaaatattttaagcaaaataatatttttatctataatctctatatggattattctagattttggggaccctatataaaattagctatgagtattaacacttgataatcttgcctagaatgtcttcgatttctgggtctaccactacacctaactgagtttaaccctgcaataaataattaatctcgtgaaatcatttggagattttgactcaatttaaataggtagctactgtgtagttaggttgaaccaggacaccaggtgtaacacgagtcacatatgcatgcatgtgtattgactcaatcggccggcgcacgctatgatggtgctagaaaatgttttatacggctgtgaaaggtgtaacctgtgctgtgtcgcaaacaatatattgtttaactttgtttggccttgaactcctgggggcaaacataagatataaaagatcgatatgcctttcatgattccgtcataatctcgaccgtaattaaggcccgctctatataaacctttaaagcaaattgttagtcatataataatttattagttggattgatgcaacaaataacaactatttatttaagattaactaacttctcagttaaatttagtcactaactattagttttagaggtttggaacatgttataagaacctaaccggtctctcaacgttacaaaagctatcatatttgaacccccgtcatcggagcgcacacgttttattttgttctgttcatgtctatgctgagatactaaaattttgtgcacaagactacaaggacgagagcacctaatgaggtattaatcggttattcaaattccgtaagagttgggggtattggaagagattattagaatttttgacctgttaagatttaaacccacttaatttcttgcaacacatactgcaggtcctcagatagcgaggcgcagtcgcgcagaccgcagagcgccgaatcgtgagaaggatcagaagtgcttgttacttccgtacgggttagagcatctccaacaacgtgacctataaaaatgccctataatttgaaaatgagtatattttatagaatttagggcaccaacaaaacaccccgctccaacagtaaagccccaaatctagattatagggcagcccactacggtgtagtatatttgagtcacttgagagggtgccctatagttttttgacaaaattttatgaaatggagcactgttggagtagtttttcctgtgtagagccctatatttcaatttgaggcactagtttgaggcattgttggagatgctcttacaaatacacggaacatatttgggttcagcaacagggacggacggacggcgcgccgtgttctacagacttgccgtcgctgcttctgcatctgttcgaaaaccgtaacccccgtgcaccgctggtcagtagtcgtcgtttcgtttcgtttcgtttcgtcgcggcgatcttcgaaccgatgaagcgtggcacttggctggttggtggtggtacgccgggccagaaggtgacctgcctcgatccgaataccatgcatcgatctgtgcacgtgcctgttctcttcctactccgattaccgatagtccgggccgggaaaaagagcgcgaagccagatctgaccactaggggtctgtttggttggtttctctcgccaacctggctgtgtgagccaggatcactggagcctggctctgaagatacgaccaatctgctcgtatctggtgagcctggcccaaggtgctttaaggatcgtgcgagtctggattcaaatctacatgcagacaaccaaacacagagctcgcacgcgcatagcttggctcatagcaaccaaacagcagctacccgcatcccgcgacgcaagcacgcgcagatgcaggcaaccaaacagaccccaggtgatccaggtcgtcgaatgttccacgcgaaccgcagccgcgagtgccgtctccggctccgccacaggtagagctcagagcagaaaatgctccagccacctgtctctcttcttcctccttcctgccctgcacgcggctataagtacccaccgccatctcactttgcccagcagcacggcagtagctgcgccattgcacgcctccggccggcggctgcttgtcttgctgcttctgccgcacatccgtgatccgtacgtcgtcacctcaccactcacgcacaagcacaaagagcgttaatttcttgctggagattctattctatttctatggcgtgtccctgatcgaatcctaaatcctaaacctctgtggtgcactgcagggcagaaggtgcttcgttcgttgcaggcctggcgccacgccgtaccgtgcaagcgCGTTTGCTGATGTTCATCTAAttactgtataataatatctccgggcgaaagagctagcaatcgtcggcgggggaggaggggctcgattgctgctcaaggtgagttgtaattccttggctctggatttccctatctgttggctgttcatggatcatccaatggatggatggcgctccctgttctctacacctgcgtgctcttcttccctcgcctcgccggggtcttgtgtcagttactgtatctccctgttgattttaaaatctaagaagcaacaacaaaagatgattcaaaaaaatattcaaatttgaaggaccacaatgcgtgtgctactgctagctatgctaccattagagcatgccttcactgcattcttcttcttttgttacgagtgcttaatctcatggctcgctcccttaattcttgctaccattagagcatcttcaatactttctaaaaaaaaccacttgacaaactaatgaaatcagttggtaaactaataagtttcacgagtgactaaaaaaaagataggagctagcttctagtcctagataatgctcttatgagctttccttgtccagttgtcccaactcccaacgaacaaaaaaaaaaaggtaagaaaacacatttggcctttcttcttttttcttttcaactcaaaacgatcgctcagttacaaaaaaaaaagagagcttgcaattgcgagcgagataccaccgttacagggaaaaaaaagacaagttgttcaagttctctactagcttcctagcgcttccgtgtcgttctagatgagcttctctagcaaaggacaataatttggttgccacgtcagatgtcgactcagtgtcatttgctaccagctggcttatcaacttgggagattattgctcgcacctggacccggtgtccagtcaattattaatgatgttgatccatcttcgtatttttatcttggcaagaaactgttagtattaagttactgtcacctttggaagctgaatctcccctcgaagatatcagtatgggcatatagccatccgttcttatacacagctatttacgtctattttacaattttatatcttcgtcttcctcttttacacctacattcgaaccatctatttagagctttcaatgtgcaattcgtctttggtcattgtcaacatgaaccgtccagtgataatgctttgatgctgactaagaagtacggtctccggttcttaaatatttattgtctaatatttatttttaaaataaaacatgataaataaaaaagaacggagtgagtagaatacattgtgagctgttgttggtttgttgcacattctttacttgtttttttttacgaacatttgttgcaagcatcagcaaagccgtataaacttgtgcagctctagatagcgatttttttaaacaaaaccttaatattagattttggagcattgatttagaaagctgagcaactccaatgggagaggtgtttattttctcgtccatccacatccgccgttggcccgttgtttcttttctacccgcgcctgtggggcccaaccgtccggtcaaccgaccagcgtttcctgttccaccgtacgtcgtcgttctcgttcggtcgttttcgttcccctacctccagtccagtcccaggtccgagccggaccttgatcgccgccgtgcctcggcgcaaggaatggggccttcggtcttacccttgcacgcgccgccggcatcaggagacgtctctgtgtgcttcgccgtgccttcagccgtagccggcgccgcatcagcgtgctccagagaggaccgcagcttccagcacgtgtccctgacaccgccccacactggattgggaagggccgctgaccccacgcacctgcccgctggccagtgttggaaggtttgggaaatgagatgttgattttaagctgacttttgagggttttagcttacagctttttaaatcaatcttcgaccaacggtttgaaattccgtgtttagagttgaattactcgattcagaagtttaagtttctctaatttaagctaaagggagaagagatggagcgcctggcttgagttggccgcacgcagctgggaagaaggatctgaaaacactgtcgtccatgtattgattcacttaaacatttgtccgtatctattattttaatttttttataatctacggtcacaagatatgcctgtgttgtttgtgaatagaaaacactgaacaatgattgtgagtcaacagctatcattatttgtgttttggttgtgcgagggtatactaatgtctaatgattggctaaaccttagtcttacatcgctgtctttccttgcgctgtagggcaaggcaaccaccaattgggtaaaagcatataagcaggcttaccgatcaataaatataaaaaagggtagctttcaagaagtctgcttatgtaataccattattttccttttttttacctcgaaggagtgataatcaccaaaatagcattatattgtcatcatacggctgcactatctttttcttctgtaacatgccgtctaattattatcttcagtttcagactcagttatttgaaacatcaagATGGTTAATTTCGGAAAGAAATTGATGGCTGATCAAGTGGACGAATGGAAAGGGtatgacttttcttttggtacttatgaaatcatctatttttatcttatcagggcaaatgttctttattttcatatatgcccactccactagtccactaggatacattagaacaccataccgtagttatcaccatatcacagtccttactatcattatctggttaattttataattaaattaaacaaaatctagcagttatcgaattgataggttgcactacaataatgaagtcacttcccgctaatgcaagctaatgtcactttgtttaacggtttatcagaaatcttatcagctttttggtctttccatttctagatactacatcaattacaagaaatcttatcagctttttggtctttccatttctAGATACTACATCAATTACAAGCTGATGAAGAAAATGTTAAAGCAATATGTCCAACAAACCCAACATGATGAGAAAGATCGCGAACAAGTTCTTAAAGACTTTTCAAGGTTTCTTGATGAccaggtatacaaagaaagatttcccttgaaatgatcataatatatgattttgagcatcatctatcctgtcagtagtcacttgtatccttgtaaggaacagaacagtgtcatgcgacaagcttaatagtcttagtgaattgggatcatttttcttagttgtgagctaaaatacacatgtatttcttcgttCCAGATTGAAAGGATTGTGCTTTTTCTGCTACAACAACAAGGCCATCTTGCCAGTAGGATTGAGAAATTGGCAGAAAAACGCACTGCTCTTCTGGAAGAGTATGACATATCACAAGTTTATCAGCTGCATGATGCATACAGGGAAGTCGGGCTTGATCTCATAAAGCTTCTCCGCTTTGTTGATGTGAATGCTACTGGTATACGCAAGATACTAAAGAAATTTGATAAACGCTTTGGCTACAAGTTCACTGATTATTATGTCACCACTCGTGCAAATCATCCTTATTCTCAGCTTCAGCAAGTATTTAAGCAAGTGGtaattttcatgcattttgcattttcctttcttgatgtgtgaagtaattcccagtacctattatttatcatggactcatacggatgcaggGAATTGTAGCTGTTGTAGGTGCATTATCGCGCAACCTTGAATATCTGCAGCATCATGAAGGAAGCTTTGTATCCATCTATGATCGTCCAGCAGTTACCTTGAaggtattctattttcactattccattctcatttcagaaattctgctattgaatttataaatgaaaaccttgaaaggtgctctttcttacctcggaactgcatcaattatatttccacatgaagtagggtgtgacatgacacttttttgttgttatattcAGGACCCTATTATAGACCAAGTAAACCATGCAGTACAGAAACTCACGCATGCCACGAATTTTATGCAATTCTTGGGACAGCACGCGCTTATTGTCCAGGAAGATGCAGAAAGCGAGTCGGAGGATCTTGTTGGTGATCAGAGCTACCATTTCATGTCCCTGGTGCTTAATCTAGTGAACACATTCCTTTACATGGTGAATACATATATCATTGTGCCGACTGCAGATGACTATGCAGTAAGCCTTGGGGCTGCTGCAACTGTATGTGGTATAATTATTGGATCGATGGCAGTCGCCCAAGTATTCTCCTCAGTCTACTTCAGTGCCTGGTCAAATAAGTCCTACTTCAAACCTCTTGTGTTCAGTAGCATTATGCTGTTTCTTGGAAACCTACTGTATGCATTGGCATATGATCTGAATTCACTAATAGTTCTCCTGACTGGACGACTGCTATGTGGGTatgcaattttctcaattcactctatctcacttgatttacgttccacttttgtatgctagcattgatctgggtgaaaattactagtatgacaaatgcaggttgaggatccttaagctgagggcaatattctagaatatttatattgctgaatagaaaacaaaatggaaactgtatatcttacaaggagataaaggattttaaatctcgagactggcattaaaatatatgcttttctatttcttttatagaacttaactagttatccctacctccctttgggctagtaatttgtctatattgtttaaggttcttgattttctgacggtgcatctgtgatcgagctgccagcatgtaatgtgcaggtTAGGTTCTGCAAGAGCAGTGAACCGTCGCTATATCAGTGACTGTGTGCCTCTCAAGATGAGGCTACAAGCCTCTGCCGGGTTCGTTAGTGCTAGCGCTCTTGGCATGGCATGTGGCCCTGCTCTTGCTGGTTTTCTCCAGATTAAATTCAAGATATACTCGCTCAGTTTTAATCAGAGCACATTGCCTGGATGGGTCATGTGCATTTCTTGGCTTATTTACTTATTGTGGTTGTGGCTTACATTCAAGGAACCAGAACACTTCACTAAAACTCTGGTCAATGAACAGCCGTCAGAATCTGGtaagctaacaatacactgaaatggcaacatgttttgtttgaattcatgaatatgctcgaatcaaaccttattgtacaatcaggatgtgtatgcttatcattcttaggaacttttctgagatgtttatttccttattatgaaaataggCCGCCAAGGAAATTCTAACTTGGAGGCAGGTCTAGCTGAACCATTGCTTCAAGGTATAGAACGAAGGCAGGATGAGAACTCAGAAGTTAATGATGATACTGAAGTAGAGTCAGAAAGCTCTCATGAACCAGCAACATCAATTGCTTCAGCATACAGATTGCTAACTCCATCTGTGAAGGtttccttccccctcccttcccaattatcgatttttctttgtcttgttcttggttcaaacgtttgaaagaaagaagctcacaatctacatagggttcttttgtaaataaagttagataaattaactatataatataataaaactgcatctttaaatagtaatgggtaaatacattccagttactagtaggtacatctgcatgtcatacaagcaaacataacggacacgccatgtaaaagaaactaggccaacctaggagaaactaggttgcatttattattatattattattaagatgaaaataggcacccaaatttaccttgacaaggacttgaacctaggtggtctgggtttacgagcacacccttgaccaagtgagctagctcagttcccttgacacaccagccagatgaaaagttgcatgtgcagctacccctttttccacgcccttttcatcttccaatctttgttggagctaccttctcatgtattcatatctttaaaaaatggttgtttgtcAGGCCCAGCTACTGATATACTTCATGCTCAAGTACGCAATGGAAATACTACTATCAGAATCGAGCGTTGTCACCACATACTATTTTAGCTGGTCTACAAGTGCTGTGGCTATCTTTCTAGCGATTCTTGGATTAACGGTTCTTCCAGTAAATGCCATTGTTGGAAGCTACGTTACAAATTTATTCGAGGATAGGtaagctttgtactcttacaaaacatactacatgaaacttttatattctctagacattgtttttctcttaaactgagacatgattcacaaaataagaacttgctctatcatttgcaggCAAATTTTGTTGGCATCTGAAGTCATGGTTCTCATCGGTATAATCATGAGCTTTTGTTTCACACCTCACTACTCCATCCCGCAATATGTTCTTTCAGCTTTCATCACATTTGTATTTGCTGAGGTGCTTGAaggtatgtatgtttatatacattaatggtttgagacagcgaaacctaaatcaatcatacgctgctgatttatcagccactcaacttctaacctcgagctatgcAGGAGTGAATCTGTCCTTGCTCTCACGAGTAATGTCATCGAGGCTTTCCCGAGGGACCTACAATGGTGGACTCCTTTCGACAGAGGCCGGGACGTTGGCCCGTGTAGTTGCAGATGCCACGATTACTGCAGCCGGTTATCTCGGCACGGACCTCCTTCTGAATGTCACTCTTCTCCCATCCCTTGTGATTTGCATAGTCTCCATCGCAGCAGCACTCTACACTTACAACAATCTCTATTGAagctattgttgctgtacaagtgtacaacaatgttcctaagctaaaatgttcctgcccacaacgggtttgtatatctgttcaagcatggtttgtaaacattttgatcaagtttgtatgcaaaatttcttgtatttagtgcatttatgtaaagattcatcctgtaaagaattataaactatgagacgctattgccatttatgatcatttatgtttatctttttagccttatgttatttgaatttgtctaatcaatgccatcttccatcacgagcacatcattgacatattactgatggatagacctttttgggacgtgagatgctaaaggcacaacctatatagctgaaaatttctaacatatttatcaataataccaagatgcctttgttgtttatcaattgcacattatttcaacgtaaatgcaattttgttaaatatgtcatggtgtcgagcctactgcttaccccagcatgttgaattgctgccttaagcagaaacatcgaaaaacctgcgtagattccacgatccaacaatcctctccgttcatttttttagttccatatgaaaggaccgattacgtctgaaagaagagtttcattagacaatctatttcttttaactaatgcctctagtttttcaaaacctatgcaataaataggtgtaactatagttttgactaagggttgtaatctctttgtaaaatatttatgcaatctcgatttcaaatctatccactagcctaacaactaataagataaaacatacaaccaagatacataatataaatacgggagcttaaatacgatatacatataaactcttattgatgactccattgtttttatcgagataccaagaaagacgcaagtttctccctagtcctcattggagcccagtccgcgcgagtaccaagctctcggtcaggtaacattgtggatagcctaggttttttgccacacacaagtgggtctttagtgtagcctcttctaaatgctctctatcaaaggtggtccaggaaaagattcaaaatggggccctagcagtagtagtattactactaacaacaataatgataaataagtaaatgctcaatgtgcataaaattgatctaagaagtacttgtaaactcatgtaatgttgtaaacttatttgcttatttctttatgttcttttctcacatccgaacagattccttctagaggacatgattgtaagttaaaaaataaaatagaacactaattaaatcaaagctatcgctacttgctgagttacaaaattattaaatctatttataaaatactaccaattatgtcgtacttccaaattatcaaacaattgaatagattacaaaataatatttaatcaatagattacaaaatacctactacaaattcacaaaacaatttgtctagtttagtaattattcaaactatagttgtacatagtaattaatttggctttggtttagaccctcggccttggtgaacgacgaacaacgaggtatcctatgtgtagtcatgtatgatgcgtctaggatgtagatgcagtggccagtggcaatcctcagtcttcacgaatcaggatgaacatatggagggtggggcctcgcggaatagggggactagggta.

TIGR transcript databases were searched for SPX:MFS domain codingsequences for SPX genes. Identification of such genes supports theexistence of a conserved regulatory pathway in higher plants. PutativemiRMON18 target sites were found by searching for a conserved sequencecomplementary to miRMON18 in the 5′ untranslated region upstream of thestart codon. A putative mirMON18 target (TA40434_(—)29760) wasidentified in grape (Vitis vinifera) with the sequence of SEQ ID NO.8768, with the miRMON18 recognition site located at nucleotides 323-343of SEQ ID NO. 8768. Similarly, a miRMON18 target (TA5852_(—)4236) wasidentified in lettuce (Lactuca sativa) as having the sequence, SEQ IDNO. 8769, with the miRMON18 recognition site located at nucleotides64-84 of SEQ ID NO. 8769.

Orthologous SPX-domain-containing genes including NLA-like genes wereidentified in various species including maize, rice, and soybean. Wheresequence was available, putative miRMON18 or miR827 target sequences(recognition sites) were identified in the 5′UTR. FIG. 12 depicts thephylogenetic tree constructed for the identified SPX genes using aminoacid sequences aligned by ClustalW, with bootstrap values determinedusing 10000 iterations; genes containing a predicted miRMON18recognition site (in genes from species other than Arabidopsis thaliana)or a predicted miR827 recognition site (in genes from Arabidopsisthaliana) that has been experimentally validated are indicated in boldtext. In addition to the Arabidopsis thaliana NLA gene AtNLA containingthe SPX and RING domains (MRT3702_(—)101115C, At1g02860, SEQ ID NO.8770, which includes a miR827 recognition site at nucleotides 135-155,and encodes the protein with the sequence of SEQ ID NO. 8771), the genesincluded in the phylogenic tree are the maize NLA-like gene ZmNLA (SEQID NO. 8772, encoding the protein with the sequence of SEQ ID NO. 8773),the soybean NLA-like gene GmNLA (SEQ ID NO. 8774, encoding the proteinwith the sequence of SEQ ID NO. 8775), the rice NLA-like gene OsNLA (SEQID NO. 8776, encoding the protein with the sequence of SEQ ID NO. 8777);the Arabidopsis sequences At1g63010 (SEQ ID NO. 8778, which includes amiR827 recognition site at nucleotides 153-173, and encodes the proteinwith the sequence of SEQ ID NO. 8779), AT2g26660 (SEQ ID NO. 8780,encoding the protein with the sequence of SEQ ID NO. 8781), andAT5g20150 (SEQ ID NO. 8782, encoding the protein with the sequence ofSEQ ID NO. 8783); the rice sequences Os02g45520 (SEQ ID NO. 8784, whichincludes a miRMON18 recognition site at nucleotides 395-415, and encodesthe protein with the sequence of SEQ ID NO. 8785) and Os04g48390 (SEQ IDNO. 8786, which includes a miRMON18 recognition site at nucleotides334-354, and encodes the protein with the sequence of SEQ ID NO. 8787);and the maize sequences MRT4577_(—)36529C (SEQ ID NO. 8788, whichincludes a miRMON18 recognition site at nucleotides 1660-1680, andencodes the protein with the sequence of SEQ ID NO. 8789),MRT4577_(—)51705C (SEQ ID NO. 8790, encoding the protein with thesequence of SEQ ID NO. 8791), MRT4577_(—)375264C (SEQ ID NO. 8792,encoding the protein with the sequence of SEQ ID NO. 8793),MRT4577_(—)46983C (SEQ ID NO. 8794, encoding the protein with thesequence of SEQ ID NO. 8795), MRT4577_(—)340665C (SEQ ID NO. 8796,encoding the protein with the sequence of SEQ ID NO. 8797), andMRT4577_(—)319995C (SEQ ID NO. 8798, encoding the protein with thesequence of SEQ ID NO. 8799).

Several kilobases of sequence were available upstream of the maize andrice NLA-like coding sequence, within which a miRMON18 target site wasnot identified from preliminary sequencing efforts. Based on expressionprofiling data it appears that ZmNLA (SEQ ID NO. 8772) RNA level doesnot respond to nitrogen availability. In contrast, the SPX-MFS domainClade (SEQ ID NO. 8788, SEQ ID NO. 8786, SEQ ID NO. 8784, and SEQ ID NO.8778) shown in FIG. 12 is suppressed under sufficient nitrogen in maizeand Arabidopsis and is predicted to be regulated by miRMON18; sequencesin this clade contain an experimentally validated miRMON18 (or miR827)recognition site. The SPX clade shown at the top of the tree (SEQ ID NO.8798, SEQ ID NO. 8796, SEQ ID NO. 8794, SEQ ID NO. 8782, SEQ ID NO.8780, SEQ ID NO. 8792, and SEQ ID NO. 8790) (FIG. 12) contains only anidentifiable SPX domain, is suppressed by limiting nitrogen, and is alsopredicted to be regulated by miRMON18. In maize, the T×P datacorresponding to genes in clade 1 (SEQ ID NO. 8798, SEQ ID NO. 8796, SEQID NO. 8794, SEQ ID NO. 8792, and SEQ ID NO. 8780) were correlated tomiRMON18 precursor expression; expression of both the miRMON18 precursorand of clade 1 genes was upregulated under conditions of nitrogensufficiency. Genes in the SPX-MFS clade 2 (SEQ ID NO. 8788, SEQ ID NO.8786, SEQ ID NO. 8784, and SEQ ID NO. 8778) are directly suppressed bymiRMON18, whereas clade 1 genes lack a direct target, suggesting thatmiRMON18 may indirectly regulate clade 1 gene expression throughsuppression of clade 2 genes.

Example 8

This example describes identification of a crop plant miRNA (miRMON18)promoter having an expression pattern characterized by strong expressionunder nitrogen-sufficient conditions and suppression undernitrogen-deficient conditions, or strong expression underphosphate-sufficient conditions and suppression underphosphate-deficient conditions

Further characterization of the maize miRMON18 gene involved BLASTmatching of a miRMON18 precursor (SEQ ID NO. 3936) to cDNA libraries andmicroarray elements, and inverse PCR cloning of a miRMON18 genomicsequence (SEQ ID NO. 8800) from maize (Zea mays var. LH244) usinginverse PCR primers based on a cDNA sequence (SEQ ID NO. 8801) fromclone LIB5025-018-A1-XP1-B6. This miRMON18 genomic sequence (SEQ ID NO.8800) had the annotated sequence depicted in FIG. 13, where the miRMON18transcript is given in upper-case text at nucleotides 2173-2788 of SEQID NO. 8800. This genomic sequence also included a miRMON18 promoterelement in lower-case text at nucleotides 211-2172 of SEQ ID NO. 8800, aleader element in lower-case text at nucleotides 2173-2308 of SEQ ID NO.8800, a canonical TATA box (ending 25 nucleotides upstream of thetranscription start site) in underlined lower-case text at nucleotides2144-2147 of SEQ ID NO. 8800, the mature miRMON18 as underlinedupper-case text at nucleotides 2419-2439 of SEQ ID NO. 8800, and themiRMON18* as underlined upper-case text at nucleotides 2322-2341 of SEQID NO. 8800.

To verify the expression pattern of the miRMON18 promoter, tworecombinant DNA constructs (SEQ ID NO. 8802 and SEQ ID NO. 8803) wereconstructed in a binary vector that included a rice actin 1 promoterdriving neomycin phosphotransferase II (nptII) as a selectable marker.The construct (SEQ ID NO. 8802) in plasmid pMON111971 included amiRMON18 promoter (SEQ ID NO. 8804) and a miRMON18 leader sequence (SEQID NO. 8805) driving expression of a GUS gene (SEQ ID NO. 8806) followedby a NOS terminator sequence (SEQ ID NO. 8807). The construct (SEQ IDNO. 8803) in plasmid pMON111967 contained a DnaK intron (SEQ ID NO.8808) and also included a miRMON18 promoter (SEQ ID NO. 8804), amiRMON18 leader sequence (SEQ ID NO. 8805), a GUS gene (SEQ ID NO. 8806)followed by a NOS terminator sequence (SEQ ID NO. 8807). The vectors aretransformed into maize using Agrobacterium-mediated transformation andantibiotic selection using standard techniques as described under theheading “Making and Using Non-natural Transgenic Plant Cells andNon-natural Transgenic Plants”. Strong miRMON18-promoter-drivenexpression of GUS is observed in transformed maize leaves undernitrogen-sufficient and phosphate-sufficient conditions. GUS expressionis suppressed in the transformed maize leaves under nitrogen-deficientor phosphate-deficient conditions.

Alternative miRMON18 promoter sequence useful for driving expression ofa transgene with the expression pattern of the native miRMON18 gene(i.e., strong expression under nitrogen-sufficient conditions andsuppression under nitrogen-deficient conditions, or strong expressionunder phosphate-sufficient conditions and suppression underphosphate-deficient conditions) include the promoter having the sequenceof nucleotides 211-2172 of SEQ ID NO. 8800; a fragment of at least about50, at least about 100, at least about 150, at least about 200, at leastabout 300, at least about 400, or at least 500 contiguous nucleotideshaving at least 85%, at least 90%, at least 95%, or at least 98%identity to nucleotides 211-2172 of SEQ ID NO. 8800, wherein thefragment has promoter activity in at least one plant tissue that ischaracterized by strong expression under nitrogen-sufficient conditionsand suppression under nitrogen-deficient conditions or strong expressionunder phosphate-sufficient conditions and suppression underphosphate-deficient conditions; and a fragment of at least about 50, atleast about 100, at least about 150, at least about 200, at least about300, at least about 400, or at least 500 contiguous nucleotides havingat least 85%, at least 90%, at least 95%, or at least 98% identity toSEQ ID NO. 8804, wherein the fragment has promoter activity in at leastone plant tissue that is characterized by strong expression undernitrogen-sufficient conditions and suppression under nitrogen-deficientconditions or strong expression under phosphate-sufficient conditionsand suppression under phosphate-deficient conditions. Identification ofalternative promoter sequences is confirmed by routine techniques, suchas verification of a TATA box within the promoter sequence andvalidation of promoter activity in at least one plant tissue (e.g., bytesting a recombinant DNA construct including the promoter drivingexpression of a reporter gene such as GUS or luciferase in eithertransient expression experiments or in stably transformed plants).

Example 9

This example describes identification of recognition sites of a cropplant miRNA (miRMON18) having an expression pattern characterized bystrong expression under nitrogen-sufficient conditions and suppressionunder nitrogen-deficient conditions, or strong expression underphosphate-sufficient conditions and suppression underphosphate-deficient conditions. Also disclosed are methods of use of themiRNA, the miRNA promoter, and a miRNA recognition site. Non-limitingexamples including a method of providing a non-natural transgenic cropplant having improved yield under nitrogen or phosphate deficiency byexpressing in the transgenic crop plant a recombinant DNA constructincluding a miRMON18-unresponsive transgene, and a method of providing anon-natural transgenic crop plant having improved yield under nitrogenor phosphate deficiency by expressing in the transgenic crop plant arecombinant DNA construct including a miRMON18 recognition site that hasbeen added to the sequence of a normally miRMON18-unresponsive gene.

Prediction of a recognition site is achieved using methods known in theart, such as sequence complementarity rules as described by Zhang (2005)Nucleic Acids Res., 33:W701-704 and by Rhoades et al. (2002) Cell,110:513-520. One non-limiting method to experimentally validatepredicted miRNA recognition sites is the technique known as RNAligase-mediated rapid amplification of cDNA 5′ ends (“5′ RLM-RACE”),which identifies miRNA cleavage patterns; see, for example, Kasschau etal. (2003) Dev. Cell, 4:205-217, and Llave et al. (2002) Science,297:2053-2056. This approach relies on ligation of an RNA adaptermolecule to the 5′ end of the cleavage site and is dependent on the 5′phosphate left by RNase III enzymes including Ago1. The resulting PCRproducts are sequenced and the relative number of clones which align tothe predicted miRNA cleavage site between nucleotides 10 and 11 relativeto the miRNA 5′ end provide an estimate of miRNA activity. FIG. 14depicts the predicted cleavage by miRMON18 of the rice sequencesOs02g45520 (SEQ ID NO. 8784) and Os04g48390 (SEQ ID NO. 8786) and themaize sequence MRT4577_(—)36529C (SEQ ID NO. 8788). Results from 5′RLM-RACE assays were used to confirm cleavage of the predicted miRMON18recognition sites (target sites) in the rice sequences Os02g45520 (SEQID NO. 8784) and Os04g48390 (SEQ ID NO. 8786), for which 3 of 24 clonesand 13 of 16 clones, respectively, were sequenced and found to have thepredicted cleavage pattern (between nucleotides 10 and 11 relative tothe miRMON18 5′ end). 5′-RACE experiments also partially validated themiRMON18 recognition site in the maize SPX_MFS2 sequence SEQ ID NO. 8767(data not shown).

Another non-limiting method to experimentally validate predicted miRNArecognition sites is to examine expression levels of the putativetarget, e.g., by transcription profiling experiments. The expressionlevel of a true target of a miRNA would be predicted to be high when themiRNA is not expressed, and low when the miRNA is expressed. Thus, amiRMON18 target would be predicted to have higher expression whenmiRMON18 is not expressed (i.e., under nitrogen-deficient orphosphate-deficient conditions), and low expression when miRMON18 isexpressed (i.e., under nitrogen-sufficient and phosphate-sufficientconditions). FIG. 15B depicts expression profiles of the maize sequenceMRT4577_(—)36529C (SEQ ID NO. 8788), which contains a predicted miRMON18recognition site. MRT4577_(—)36529C (SEQ ID NO. 8788) was unaffected bywater availability or temperature (with no large differences intranscript levels seen between water sufficient or drought conditions orbetween cold or normal temperatures; data not shown), but exhibitedhigher expression levels under nitrogen-deficient conditions than undernitrogen-sufficient conditions, i.e., an expression pattern opposite tothat of miRMON18 as shown in FIG. 15A (also see FIG. 9 and FIG. 10),indicating that MRT4577_(—)36529C (SEQ ID NO. 8788) is indeed regulatedby miRMON18.

These data verify that miRMON18 regulates conservedSPX-domain-containing genes. Expression of miRMON18 is suppressed duringnitrogen deficiency or phosphate-deficiency, allowing the endogenousmiRMON18-regulated genes to be expressed under these conditions.Manipulating the expression of either the mature miRMON18 miRNA or ofmiRMON18 targets (genes including at least on miRMON18 recognition site)is useful in altering a plant's response to nitrogen deficiency orphosphate deficiency.

One aspect of this invention includes a method of providing anon-natural transgenic crop plant having improved yield under nitrogenor phosphate deficiency by expressing in the transgenic crop plant amiRMON18-unresponsive transgene. One embodiment is expressing in anon-natural transgenic crop plant a recombinant DNA construct comprisinga synthetic miRMON18-unresponsive transgene sequence, wherein thesynthetic miRMON18-unresponsive transgene sequence is: (a) derived froma natively miRMON18-responsive sequence by deletion or modification ofall native miRMON18 miRNA recognition sites within the nativelymiRMON18-responsive sequence (that is to say, eliminating or changingnucleotides of the natively miRMON18-responsive sequence that arerecognized by a mature miRMON18 miRNA having the sequence of SEQ ID NO.393, SEQ ID NO. 3227, or SEQ ID NO. 8742 or by a mature miRMON18 miRNAderived from a miRMON18 precursor sequence selected from SEQ ID NO.1763, SEQ ID NO. 3936, and SEQ ID NO. 8800), and (b) is not recognizedby a mature miRMON18 miRNA. In a non-limiting example, the miRMON18recognition site in any of the conserved SPX-domain-containing genesdepicted in FIG. 12 is deleted or modified such that the modified SPXgene is not recognized and bound by an endogenous mature miRMON18 (SEQID NO. 393, SEQ ID NO. 3227, or SEQ ID NO. 8742) and is therebydecoupled from miRMON18 regulation and thus from the influence ofnitrogen or phosphate deficiency. In a non-limiting specific example,the maize gene MRT4577_(—)36529C (SEQ ID NO. 8788) can be decoupled frommiRMON18 regulation and thus from the influence of nitrogen or phosphatedeficiency by expression of a modified MRT4577_(—)36529C gene whereinthe miRMON18 recognition site in its 5′ untranslated region (nucleotides1660-1680 of SEQ ID NO. 8788) has been deleted or modified such that themodified MRT4577_(—)36529C gene is not recognized and bound by a maturemiRMON18 (SEQ ID NO. 393, SEQ ID NO. 3227, or SEQ ID NO. 8742). Themodified miRMON18-unresponsive MRT4577_(—)36529C is expressed usingconstitutive or tissue-specific promoters in maize, increasing nutrientuptake and utilization under nitrogen-sufficient andphosphate-sufficient conditions and resulting in improved yield undernormal conditions and preferably also under nitrogen-deficient orphosphate-deficient conditions.

Alternatively, the miRMON18 recognition site is engineered into normallymiRMON18-unresponsive genes that are to be suppressed undernitrogen-sufficient conditions and expressed during nitrogen-deficientconditions; this is a useful approach, e.g., with a nitrogen-transportgene that gives increased performance or yield when expressed undernitrogen- or phosphate-limiting conditions, but provides no benefit whenexpressed under non-limiting conditions.

Example 10

Additional non-limiting examples of methods and recombinant DNAconstructs useful in improving nitrogen or phosphate utilization basedon manipulating miRMON18 or SPX gene expression are described below.

(A) Modulation of SPX Gene Expression to Improve Nitrogen UtilizationUnder Limiting Conditions.

In this embodiment, a SPX-domain-containing gene engineered to lack amiRMON18 recognition site (or in Arabidopsis thaliana, a miR827recognition site) in the 5′ UTR is expressed in plants. Decoupling theSPX gene from endogenous miRMON18 (or in Arabidopsis thaliana, miR827)regulation provides adaptation to nutrient availability under nitrogen-or phosphate-sufficient conditions, and result in increased yield. Onedesirable result of increasing expression of the SPX-MFS clade (SEQ IDNO. 8788, SEQ ID NO. 8786, SEQ ID NO. 8784, and SEQ ID NO. 8778) (FIG.12) is the increase of protein content in at least one plant tissue(such as leaf, stalk, root, or seed) during nitrogen- orphosphate-sufficient conditions through increasing nutrient availabilityin sink tissues. Several vectors (see Table 7) are evaluated inArabidopsis thaliana to model the function of SPX genes.

The predicted phenotype of upregulating AtNLA (At1g02860, containing anSPX-RING domain, SEQ ID NO. 8745) in a plant is constitutive adaptationto low nitrogen or low phosphate conditions and improvement of overalltransport and utilization of nutrients by the plant; a similar phenotypeis predicted for upregulating the related genes in the SPX-RING clade(SEQ ID NO. 8772, SEQ ID NO. 8770, SEQ ID NO. 8774, and SEQ ID NO. 8776)(FIG. 12). Vector numbers 1 through 5 are chimeric transcripts includingnon-targeted 5′ and 3′ untranslated regions and the coding region ofAtNLA (SEQ ID NO. 8745); vector number 6 includes a genomic AtNLA (SEQID NO. 8745) fragment including the sequence from the endogenous AtNLApromoter through the termination sequence (to prevent ectopicexpression) but where the native miR827 recognition site has beendeleted or modified to prevent recognition by a mature miR827.

The predicted phenotype of upregulating SPX-MFS clade genes (SEQ ID NO.8788, SEQ ID NO. 8786, SEQ ID NO. 8784, and SEQ ID NO. 8778) (FIG. 12)is increased nutrient (especially nitrogen and/or phosphate) transport,particularly from source to sink tissues, resulting in increased yield.Vector numbers 7, 9, and 10 are chimeric transcripts includingnon-targeted 5′ and 3′ untranslated regions and the coding region ofAt1g63010 (SEQ ID NO. 8778); vector number 8 includes a genomicAt1g63010 (SEQ ID NO. 8778) fragment including the sequence from theendogenous At1g63010 promoter through the termination sequence (toprevent ectopic expression) but where the native miR827 recognition sitehas been deleted or modified to prevent recognition by a mature miR827;vector number 11 is a chimeric transcript including non-targeted 5′ and3′ untranslated regions and the coding region of Os04g48390 (SEQ ID NO.8786); vector number 12 includes a genomic Os04g48390 (SEQ ID NO. 8786)fragment including the sequence from the endogenous Os04g48390 promoterthrough the termination sequence (to prevent ectopic expression) butwhere the native miRMON18 recognition site has been deleted or modifiedto prevent recognition by a mature miRMON18 (or in Arabidopsis thaliana,by a mature miR827).

The effects of upregulating genes from the SPX clade of unclassifiedfunction but predicted to be repressed by low nitrogen availability isevaluated by expression of MRT4577_(—)319995C (SEQ ID NO. 8798) withvectors 13-15 (Table 7).

Similar expression experiments are conducted in maize. Vectors (Table 7)including genes with a conserved SPX domain (see FIG. 12) areconstructed and transformed into maize. Vector variants include a vectorincluding the SPX gene's genomic sequence and vectors including the SPXgene's cloned cDNA driven by the native promoter. In non-limitingexamples, vectors 16-18 use the coding sequence from MRT4577_(—)36529C(SEQ ID NO. 8788), and have a disrupted miRMON18 recognition site,permitting expression of the transgene under sufficient nitrogen onlywhere the native promoter is expressed. A third construct to expressonly the MFS domain from MRT4577_(—)36529C (SEQ ID NO. 8788), whichrepresents a native alternatively spliced isoform lacking SPX, will alsobe made and tested for nitrogen assimilation in maize.

TABLE 7 Vector Predicted expression No. Construct* pattern 1 35S: AtNLAconstitutive 2 35S: AtNLA lacking SPX domain constitutive 3 35S: AtNLAlacking RING domain constitutive 4 FDA/PPDK: AtNLA leaf-specific 5 RCc3:AtNLA root-specific 6 AtNLA promoter: AtNLA with native AtNLA disruptedmiR827 recognition site expression 7 35S: At1g63010 constitutive 8At1g63010 promoter: At1g63010 with native At1g63010 disrupted miR827recognition site expression 9 35S: At1g63010 lacking SPX domainconstitutive 10 35S: At1g63010 lacking MFS domain constitutive 11 35S:Os04g48390 constitutive 12 Os04g48390 promoter: Os04g48390 withdisrupted miRMON18 recognition site 13 35S: MRT4577_319995C constitutive14 FDA/PPDK: MRT4577_319995C leaf-specific 15 RCc3: MRT4577_319995Croot-specific 16 Promoter ZmSPXMFS: ZmSPXMFS cDNA: Terminator ZmSPXMFS17 Promoter ZmSPXMFS: ZmSPXMFS gDNA: Terminator ZmSPXMFS 18 PromoterZmSPXMFS: ZmMFS cDNA: Terminator ZmSPXMFS *35S, cauliflower mosaic virus(CaMV) 35S promoter; FDA, fructose bisphosphate aldolase promoter(bundle sheath promoter); PPDK, pyruvate orthophosphate dikinasepromoter (mesophyll cell promoter); RCc3, promoter of rice RCc3 gene(root specific promoter)

(B) Gene Expression Under Sufficient Nitrogen Utilizing a MIRMON18Promoter.

In this embodiment, a miRMON18 promoter is utilized to eliminateundesirable phenotypes (off-types) resulting from expression oftransgenes under limiting nitrogen. For example, when nitrogen is notlimiting the expression of asparagine synthetase gives a desirablehigh-protein phenotype. Under limiting nitrogen, overexpression ofasparagine synthetase causes a yield reduction. Expression of asparaginesynthetase driven by the MIRMON18 promoter gives a high-proteinphenotype under sufficient nitrogen availability, yet under limitingnitrogen the transgene is turned off preventing the yield penalty.Vectors are constructed including the maize miRMON18 promoter (SEQ IDNO. 8804), maize miRMON18 leader sequence (SEQ ID NO. 8805), and amiRMON18 foldback structure fused to an asparagine synthetase gene.Non-limiting examples of an asparagine synthetase gene include a soybean(Glycine max) asparagine synthetase (SEQ ID NO. 8809), a Galdieriasulphuraria asparagine synthetase (SEQ ID NO. 8810), and a maize (Zeamays) asparagine synthetase (SEQ ID NO. 8811). These vectors aretransformed into maize, and yield and protein quality are evaluated inthe resulting transgenic maize plants under limiting and sufficientnitrogen.

(C) Gene Suppression Under Limiting Nitrogen Utilizing a miRMON18Recognition Site Sequence. A non-limiting example of this embodiment isa recombinant DNA construct including a transgene transcription unit andan exogenous miRMON18 recognition site, wherein expression of therecombinant DNA construct in a plant results in expression of thetransgene when the mature miRMON18 miRNA is not expressed. The 5′UTR ofSPX-domain-containing genes of higher plants confers suppression of themRNA under sufficient nitrogen through regulation by an endogenousmature miRMON18 or miR827. In a non-limiting embodiment of thisinvention the 5′UTR of an SPX gene regulated by miRMON18 or miR827, suchas, but not limited to, AtNLA (SEQ ID NO. 8770), At1g63010 (SEQ ID NO.8778), Os02g45520 (SEQ ID NO. 8784), Os04g48390 (SEQ ID NO. 8786), andMRT4577_(—)36529C (SEQ ID NO. 8788), is incorporated in the leadersequence of a transgene expression cassette. This results in suppressionof the transgene under sufficient nitrogen, regardless of promotersequence utilized, to eliminate off-types associated with unregulatedtransgene expression. In a preferred embodiment, the conserved4-nucleotide sequence AUG(G/U) present at the cleavage site in themiRMON18 or miR827 recognition site is changed to GUGG to preventunintended initiation while preserving base-pairing to the mature miRNA.Alternatively, synthetic miRMON18 or miR827 recognition sites areincorporated into non-translated regions, or within the coding regionwithout changing the protein function, to confer suppression undersufficient nitrogen.

In another example, the 5′ UTR of Os04g48390 (SEQ ID NO. 8786) is fusedto GUS driven by a constitutive promoter; one version containing theendogenous rice sequence with AUG present at the miRMON18 cleavage site,and another version wherein the AUG at the miRMON18 cleavage site hasbeen modified to GUG are constructed. A third construct, with tandem(two or more) synthetic miRMON18 recognition sites introduced into the3′ UTR is also evaluated. These vectors are evaluated in transformedmaize plants grown under varying nutrient (nitrogen or phosphate)conditions and various tissues assayed for GUS expression.

(D) Ectopic Expression of MIRMON18 to Limit SPX Gene Expression.

In this embodiment, miRMON18 (or in Arabidopsis, miR827) expression isdriven by a constitutive or tissue-specific promoter, resulting insuppression of all miRMON18-regulated (or miR827-regulated) genes suchas the conserved SPX genes. One non-limiting example includes the vectorpMON107261 (FIG. 16), which includes a CaMV 35S promoter drivingexpression of the maize miRMON18 transcript (e.g., nucleotides 2173-2788of SEQ ID NO. 8800). Phenotypes of transgenic maize and Arabidopsisplants transformed with this vector are evaluated and plants exhibitingimproved traits such as increased yield under nitrogen- orphosphate-limited conditions are selected.

Example 11

This example describes a recombinant DNA construct that is transcribedto an RNA transcript including at least one miRNA decoy sequence that isrecognized and bound by an endogenous mature miRNA but not cleaved. Inone preferred embodiment of this invention, the endogenous mature miRNAis one that is responsive to nutrient stress—e.g., a mature miRNA withexpression that is either upregulated or downregulated by conditions ofnutrient deficiency, relative to expression under nutrient sufficiency.More specifically, this example describes miRNA decoy sequences formature miRNAs (miR827, miRMON18, and miR399) that are responsive tonutrient stress.

Examples 6-10 describe two miRNAs, miR827 (SEQ ID NO. 8744) and miRMON18(SEQ ID NO. 393, SEQ ID NO. 3227, or SEQ ID NO. 8742) that exhibit anexpression pattern characterized by regulation of the miRNA by nutrientstress (for example, suppression of the miRNA under conditions ofnitrogen deficiency, phosphate deficiency, or both nitrogen andphosphate deficiency). Another miRNA, miR399, identified in Arabidopsisthaliana, has the sequence UGCCAAAGGAGAGUUGCCCUG (SEQ ID NO. 8812); anidentical miRNA was identified by small RNA sequencing in maize (SEQ IDNO. 8813) rice (SEQ ID NO. 8814), and soybean (SEQ ID NO. 8815).

The maize miR399 gene was found to be responsive to nitrogenavailability. Maize miR399 precursors were identified from proprietarycDNA datasets and included a Zm-miR399 cDNA sequence(MRT4577_(—)22484C.8) having the sequence of SEQ ID NO. 8816, whichcontained a Zm-miR399 precursor (SEQ ID NO. 8817) at nucleotides 71-175of SEQ ID NO. 8816, and another Zm-miR399 cDNA sequence(MRT4577_(—)22487C.6) having the sequence of SEQ ID NO. 8818, whichcontained a Zm-miR399 precursor (SEQ ID NO. 8819) at nucleotides 136-330of SEQ ID NO. 8818. The fold-back structures of the maize miR399precursors are depicted in FIG. 17A; FIG. 17B depicts results oftranscriptional profiling experiments with probe A1ZM033468_atcorresponding to MRT4577_(—)22487C.6 (SEQ ID NO. 8818), whichdemonstrate that the Zm-miR399 pri-miRNA (SEQ ID NO. 8817) is suppressedunder nitrogen-deficient conditions (black bars) and is expressed undernitrogen-sufficient conditions (white bars).

In Arabidopsis thaliana, miR399 has been reported to be responsive toinorganic phosphate availability and to suppress a clade of genesincluding the Arabidopsis thaliana PHO2 gene (At2g33770, encoding an E2conjugase) and putative PHO2 orthologues from various plants. Inorganicphosphate deprivation induces expression of miR399; overexpression ofmiR399 in phosphate-replete conditions represses PHO2 expression andleads to high leaf phosphate concentrations. See Fujii et al. (2005)Curr. Biol., 15: 2038-2043; Chiou et al. (2006) Plant Cell, 18:412-421;Aung et al. (2006) Plant Physiol. 141:1000-1011; and Bari et al. (2006)Plant Physiol., 141:988-999.

A conserved 23-nucleotide motif found in the Arabidopsis thaliana IPS 1transcript and other members of the Mt4-TPSI family of genes wasreported to have a sequence complementary to miR399 except for amismatched loop corresponding to positions 10 and 11 in the maturemiR399, which prevents cleavage of the miR399:IPS1 duplex; seeFranco-Zorrilla et al. (2007) Nature Genetics, 39:1033-1037. A similarnon-cleavable sequence that also contains mismatches corresponding topositions 10 and 11 in the mature miRNA has been reported for miR390;see Axtell et al. (2006) Cell, 127:565-577.

Rules were developed for predicting an endogenous “microRNA decoysequence”, i.e., a sequence that can be recognized and bound by anendogenous mature miRNA resulting in base-pairing between the miRNAdecoy sequence and the endogenous mature miRNA, thereby forming acleavage-resistant RNA duplex that is not cleaved because of thepresence of mismatches between the miRNA decoy sequence and the maturemiRNA. In general, these rules define (1) mismatches that are required,and (2) mismatches that are permitted but not required. Mismatchesinclude canonical mismatches (e.g., G-A, C-U, C-A) as well as G::Uwobble pairs and indels (nucleotide insertions or deletions).

Required mismatches include: (a) at least 1 mismatch between the miRNAdecoy sequence and the endogenous mature miRNA at positions 9, 10, or 11of the endogenous mature miRNA, or alternatively, (b) 1, 2, 3, 4, or 5insertions (i.e., extra nucleotides) at a position in the miRNA decoysequence corresponding to positions 9, 10, or 11 of the endogenousmature miRNA. In preferred embodiments, there exists either (a) at least1 mismatch between the miRNA decoy sequence and the endogenous maturemiRNA at positions 10 and/or 11 of the endogenous mature miRNA, or (b)at least 1 insertion at a position in the miRNA decoy sequencecorresponding to positions 10 and/or 11 of the endogenous mature miRNA.

Mismatches that are permitted, but not required, include: (a) 0, 1, or 2mismatches between the miRNA decoy sequence and the endogenous maturemiRNA at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of the endogenousmature miRNA, and (b) 0, 1, 2, or 3 mismatches between the miRNA decoysequence and the endogenous mature miRNA at positions 12 through thelast position of the endogenous mature miRNA (i.e., at position 21 of a21-nucleotide mature miRNA), wherein each of the mismatches at positions12 through the last position of the endogenous mature miRNA is adjacentto at least one complementary base-pair (i.e., so that there is not morethan 2 contiguous mismatches at positions 12 through the last positionof the endogenous mature miRNA). In preferred embodiments, there existno mismatches (i.e., there are all complementary base-pairs) atpositions 1, 2, 3, 4, 5, 6, 7, and 8 of the endogenous mature miRNA.

These rules were employed to identify from proprietary cDNA datasets anumber of maize sequences or soybean sequences containing endogenousmiRNA decoy sequences. Table 8 provides maize (Zea mays) endogenousmiRNA decoy sequences for miRMON18 (SEQ ID NO. 393, SEQ ID NO. 3227, orSEQ ID NO. 8742); mismatches in the miRNA decoy sequence are indicatedby underlined text in the alignment between the miRNA and the miRNAdecoy sequence.

TABLE 8 maize cDNA identifier  Alignment between miRMON18 and SEQ ID NO. given in 3′ to 5′ direction  SEQ   (nucleotide position (above) and miRNA decoy se- ID of encoded miRNA  quence given in 5′to 3′ NO. miRMON18 decoy sequence decoy sequence in cDNA)direction (below) 8820 AGGUUGCUGAUGAAGUCAUCUAA MRT4577_321885C.1ACAAACGACUAC--CAGUAGAUU (SEQ ID NO. 8821) AGGUUGCUGAUGAAGUCAUCUAA(182-204) 8822 UCUUUGCAGAGUGUCAUCUAA MRT4577_531852C.2ACAAACGACUACCAGUAGAUU (SEQ ID NO. 8823) UCUUUGCAGAGUGUCAUCUAA (198-218)8824 UGUUUGAUAGAGAUCAUCUAA MRT4577_606578C.1 ACAAACGACUACCAGUAGAUU(SEQ ID NO. 8825) UGUUUGAUAGAGAUCAUCUAA (65-85)

Table 9 provides maize (Zea mays) endogenous miRNA decoy sequences formiR399 (SEQ ID NO. 8812, SEQ ID NO. 8813, SEQ ID NO. 8814, or SEQ ID NO.8815); mismatches in the miRNA decoy sequence are indicated byunderlined text in the alignment between the miRNA and the miRNA decoysequence.

TABLE 9 maize cDNA identifier and SEQ ID NO.Alignment between miR399 given SEQ (nucleotide position  in 3′ to 5′direction (above) ID of encoded miRNA decoyand miRNA decoy sequence given  NO. miR399 decoy sequencesequence in cDNA) in 5′ to 3′ direction (below) 8826UAGGGCAACUUGUAUCCUUUGGCA MRT4577_47862C.7 GUCCCGUUGAG---AGGAAACCGU(SEQ ID NO. 8827) UAGGGCAACUUGUAUCCUUUGGCA (699-722) 8828CAGGGCAAGUUGAAUCCUUUGGCA MRT4577_36567C.8 GUCCCGUUGAG---AGGAAACCGU(SEQ ID NO. 8829) CAGGGCAAGUUGAAUCCUUUGGCA (746-769) 8830UAGGGCAACUUGUAUCCUUUGGCA MRT4577_521786C.1 GUCCCGUUGAG---AGGAAACCGU(SEQ ID NO. 8831) UAGGGCAACUUGUAUCCUUUGGCA (156-179) 8832UAGGGCACCUUGUCUCCUUUGGCA MRT4577_135578C.1 GUCCCGUUGU---GAGGAAACCGU(SEQ ID NO. 8833) UAGGGCACCUUGUCUCCUUUGGCA (185-208)

MicroRNA miR399 decoy sequences were identified in the minus strand oftwo cDNA sequences (SEQ ID NO. 8831 and SEQ ID NO. 8833). A six-frametranslation analysis of the cDNA sequences provided in Table 9 did notreveal any long open reading frames, and BLAST searches of these samesequences did not identify any protein in public databases, indicatingthat these genes are likely non-coding sequences. Alignment of the maizecDNA sequences of the miR399 decoy sequences is depicted in FIG. 18 withthe consensus sequence given as SEQ ID NO. 8834, and reveals at leasttwo groups of genes containing miR399 decoy sequences: the first groupcontains closely related genes MRT4577_(—)47862C.7 (SEQ ID NO. 8827),MRT4577_(—)521786C.1 (SEQ ID NO. 8831), and MRT4577_(—)135578C.1 (SEQ IDNO. 8833), and the second group contains MRT4577_(—)36567C.8 (SEQ ID NO.8829). There was only a 1-nucleotide difference between the miRNA decoysequence in the first group of maize genes containing a miR399 decoysequence (SEQ ID NO. 8827, SEQ ID NO. 8831, and SEQ ID NO. 8833) and theArabidopsis thaliana IPS1 (AT3G09922.1) miR399 “mimic” site reported byFranco-Zorrilla et al. (2007) Nature Genetics, 39:1033-1037; theconserved G at position 12 of SEQ ID NO. 8826, SEQ ID NO. 8830, and SEQID NO. 8832 is replaced by a C in the Arabidopsis miR399 “mimic” site.However, homology between the maize genes (SEQ ID NO. 8827, SEQ ID NO.8831, and SEQ ID NO. 8833) and the Arabidopsis thaliana IPS1(AT3G09922.1) gene was limited to the miRNA decoy sequence site.

Table 10 provides soybean (Glycine max) endogenous miRNA decoy sequencesfor miR399 (SEQ ID NO. 8812, SEQ ID NO. 8813, SEQ ID NO. 8814, or SEQ IDNO. 8815); mismatches in the miRNA decoy sequence are indicated byunderlined text in the alignment between the miRNA and the miRNA decoysequence. Transcription profiling data was used to compare expression ofendogenous miRNA decoy cDNA sequences and the corresponding miRNAprecursors; the probeset included A1GM035741_at (corresponding to SEQ IDNO. 8836), A1GM069937_at (corresponding to SEQ ID NO. 8838),A1GM074873_at (corresponding to SEQ ID NO. 8840), A1GM031412_at(corresponding to SEQ ID NO. 8842), and A1GM053788_at (corresponding toSEQ ID NO. 8844).

TABLE 10 maize cDNA identifier Alignment between miR399 and SEQ ID NO.given in 3′ to 5′ direc- SEQ   (nucleotide position tion (above) and miRNA ID of encoded miRNA  decoy sequence given in 5′NO. miR399 decoy sequence decoy sequence in cDNA) to 3′direction (below) 8835 UAGGGCAACUUCGAUCCUUUG MRT3847_238967C. 1GUCCCGUUAAG---AGGAAACCGU GCA (SEQ ID NO. 8836) uagggcaacuucgauccuuuggca(390-413) 8837 UAGGGCAACUUCUAUCCUUUG MRT3847_241832C. 1GUCCCGUUAAG---AGGAAACCGU GCA (SEQ ID NO. 8838) uagggcaacuucuauccuuuggca(393-416) 8839 AAGGGCAACUUCAAUCCUUUG MRT3847_336885C. 1GUCCCGUUAAG---AGGAAACCGU GCA (SEQ ID NO. 8840) aagggcaacuucaauccuuuggca(96-119) 8841 AAGGGCAACUUCCAUCCUUUG MRT3847_217257C.2GUCCCGUUAAG---AGGAAACCGU GCA (SEQ ID NO. 8842) aagggcaacuuccauccuuuggca(179-202) 8843 AAGGGCAACUUCCAUCCUUUG MRT3847_236871C.3GUCCCGUUAAG---AGGAAACCGU GCA (SEQ ID NO. 8844) aagggcaacuuccauccuuuggca(238-261)

Transcription profiling experiments were used to compare expression ofmaize endogenous miR399 decoy cDNA sequences and the corresponding maizemiR399 precursors under different nitrogen conditions. Group 1 miR399decoy gene MRT4577_(—)47862C.7 (SEQ ID NO. 8827) exhibited about atwo-fold down-regulation under nitrogen-deficient conditions in maizeleaf (FIG. 19A); group 2 miR399 decoy gene MRT4577_(—)36567C.8 (SEQ IDNO. 8829) exhibited an even more dramatic down-regulation of at leastten-fold or greater under nitrogen-deficient conditions in maize leaf(FIG. 19B). These results were verified by northern blots measuringexpression of the mature miR399 (FIG. 19C) and of the miR399 decoysequence MRT4577_(—)47862C.7 (SEQ ID NO. 8827) (FIG. 19D). The northernblots were made with 5 micrograms per lane of total RNA from V6 leaffrom maize grown under low (2 millimolar) or high (20 millimolar)nitrogen, and the same blot probed for the mature 21-nucleotide miR399(FIG. 19C) and the miR399 decoy sequence MRT4577_(—)47862C.7 (SEQ ID NO.8827) (FIG. 19D, with major band at about 600 bp). Higher expressionlevels of the maize miR399 decoy sequences during nitrogen sufficiencymirror the higher expression levels of the maize miR399 precursorsduring nitrogen sufficiency (FIG. 17B).

Similar transcription profiling experiments were used to compareexpression of maize endogenous miR399 decoy cDNA sequences and thecorresponding maize miR399 precursors under different temperatureconditions. Group 2 miR399 decoy gene MRT4577_(—)36567C.8 (SEQ ID NO.8829) exhibited at least ten-fold or greater higher expression duringnitrogen-sufficient conditions in maize leaf, especially during daylighthours (FIG. 20A). This same gene exhibited at least a two-folddown-regulation in root (FIG. 20B) and in shoot (FIG. 20C) afterextended exposure to cold.

The expression of the endogenous miR399 decoy cDNA sequences were alsocompared in different tissues in both maize and soybean. FIG. 21Adepicts expression levels of the group 1 maize miR399 decoy sequence SEQID NO. 8827 (MRT4577_(—)47862C, represented by probes A1ZMO05814_at andA1ZMO05813_s_at), and the group 2 maize miR399 decoy sequence SEQ ID NO.8829 (MRT4577_(—)36567C, represented by probe A1ZM048024_at), as well asof the maize pri-miR399 sequence SEQ ID NO. 8818 (MRT4577_(—)22487C.6represented by probe A1ZM033468_at). FIG. 21B depicts expression levelsof the soybean miR399 decoy sequences SEQ ID NO. 8842(MRT3847_(—)217257C.2, represented by probe A1GM031412_at), SEQ ID NO.8844 (MRT3847_(—)236871C.2, represented by probe A1GM053788_at), SEQ IDNO. 8836 (MRT3847_(—)238967C.1, represented by probe A1GM035741_at), andSEQ ID NO. 8838 (MRT3847_(—)241832C.1, represented by probeA1GM069937_at).

These data confirm a novel nitrogen-responsive expression pattern incrop plants including maize and soybean for both the mature miR399 (andthe miR399 precursors) as well as for the endogenous miR399 decoysequences. Various utilities of the miR399 include overexpression of themature miR399 (e.g., by overexpression of a pri-miR399 sequence),expression of an engineered miR399 designed to suppress a gene otherthan one natively targeted by a native mature miR399, expression of atransgene (coding or non-coding sequence or both) under control of themiR399 promoter, expression of a transgene in which a miR399 recognitionsite has been added or removed, overexpression of a miR399 decoysequence, and suppression of an endogenous miR399 decoy sequence.

Table 11 provides soybean (Glycine max) and maize (Zea mays) endogenousmiRNA decoy sequences for miR319, UUGGACUGAAAGGAGCUCCU (SEQ ID NO.8845), which has been identified in a number of plant species includingArabidopsis thaliana, Oryza sativa, Zea mays, and Glycine max (seepublicly available examples at miRBase,microrna.sanger.ac.uk/cgi-bin/sequences/query.pl?terms=miR319);mismatches in the miRNA decoy sequence are indicated by underlined textin the alignment between the miRNA and the miRNA decoy sequence. FIG.22A depicts transcription profiling data in various soybean tissues ofthe soybean endogenous miR319 decoy SEQ ID NO. 8847(MRT3847_(—)41831C.6, represented by probe A1GM001017_at); FIG. 22Bdepicts transcription profiling data in various maize tissues of themaize endogenous miR319 decoy SEQ ID NO. 8849 (MRT4577_(—)577703C.1,represented by probe A1ZMO12886_s_at).

TABLE 11 cDNA identifier and Alignment between miR319SEQ ID NO. (nucleo- given in 3′ to 5′ direc- SEQ   tide position oftion (above) and miRNA  ID encoded miRNA decoy decoy sequence given in NO. miR319 decoy sequence sequence in cDNA) 5′ to 3′ direction (below)8846 GGGAGUUUCUACCUCCAGUCCAA MRT3847_41831C.6 UCCUCGAGGA---AAGUCAGGUU(SEQ ID NO. 8847) gggaguuucuaccuccaguccaa (545-567) 8848GGGAGCGCCAAUCAGUCCAA MRT4577_577703C.1 UCCUCGAGGAAAGUCAGGUU(SEQ ID NO. 8849) gggagcgccaaucaguccaa (751-770)

Among the target genes regulated by miR319 are the TCP genes involved inleaf development and MYB genes involved in flower development. Oneembodiment of this invention is altering a plant's leaf or floralarchitecture or developmental pattern by suppressing transcription of anendogenous mature miR319 in a transgenic plant, or to alter endogenousmiR319 activity by overexpressing a miR319 decoy sequence in atransgenic plant.

In yet another example, miR398b (SEQ ID NO. 8850) has been shown toregulate expression of CSD1 and CSD2 (copper/zinc superoxide dismutase);see Sunkar et al. (2006) Plant Cell, 18:2051-2065. Superoxide dismutaseaids in the scavenging of reactive oxygen species (ROS) by converting O₂to H₂O₂ and minimizes potential damage caused by superoxide or bysuperoxide-derived ROS. miR398 is slightly down regulated by oxidativestress and strongly downregulated by Cu availability; see Yamasaki etal. (2007) J. Biol. Chem., 282:16369-16378. One embodiment of thisinvention includes expressing an chimeric transcript including miR398bdecoy sequences (e.g., SEQ ID NOS. 8851-8852) under the control of anoxidative stress-inducible promoter, resulting in further suppression ofthe activity of miR398b and increased CSD1 and CSD2 accumulation andstress protection under stress conditions.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations can be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1-18. (canceled)
 19. A recombinant DNA construct comprising a syntheticmiRMON18-unresponsive transgene sequence, wherein said syntheticmiRMON18-unresponsive transgene sequence is selected from: (i) anSPX-domain-containing sequence wherein all native miRMON18 miRNArecognition sites within said SPX-domain-containing sequence have beendeleted; and (ii) an SPX-domain-containing sequence with modification ofall native miRMON18 miRNA recognition sites within saidSPX-domain-containing sequence, wherein said modification comprisesmismatches corresponding to positions 10 and 11 of said mature miRNA,wherein said mismatches result in prevention of recognition and cleavageby said mature miRNA; wherein said native miRMON18 miRNA recognitionsites are recognized by a mature miRNA derived from a miRMON18 precursorsequence of SEQ ID NO:1763.
 20. The recombinant DNA construct of claim19, wherein said synthetic miRMON18-unresponsive transgene sequence isnot recognized and cleaved by a mature miRMON18 miRNA having thesequence of SEQ ID NO:393, SEQ ID NO:3227, or SEQ ID NO:8742.
 21. Therecombinant DNA construct of claim 19, wherein saidSPX-domain-containing sequence comprises in its C-terminus an EXS, VTC,or MFS domain.
 22. The recombinant DNA construct of claim 19, whereinsaid SPX-domain-containing sequence comprises a nucleotide sequenceselected from: (i) SEQ ID NOs: 8756, 8758, 8760, 8762, 8764, 8767, 8768,8769, 8770, 8774, 8776, 8778, 8780, 8782, 8784, 8786, 8788, 8790, 8792,8794, 8796, and 8798; and (ii) nucleotide sequences encoding amino acidsequences selected from SEQ ID NOs: 8757, 8759, 8761, 8763, 8765, 8766,8771, 8775, 8777, 8779, 8781, 8783, 8785, 8787, 8789, 8791, 8793, 8795,8797, and
 8799. 23. A non-natural transgenic plant cell comprising arecombinant DNA construct of claim
 19. 24. A non-natural transgenicplant comprising a regenerated plant prepared from the non-naturaltransgenic plant cell of claim 23, or a progeny plant of a regeneratedplant prepared from the non-natural transgenic plant cell of claim 23,wherein said non-natural transgenic plant has improved yield undernitrogen deficiency, relative to a plant lacking said recombinant DNAconstruct.
 25. A method of providing a transgenic crop plant havingimproved yield under nitrogen deficiency, comprising expressing in saidtransgenic crop plant the recombinant DNA construct of claim 19.